On-demand nitric oxide generation and delivery device

Abstract

Apparatus and methods are provided for generating nitric oxide gas, which may be performed on demand. The method can include electrolyzing an iron (III)-ligand complex in a medium containing nitrite ions. The medium preferably has a pH of 5 to 9, wherein the iron (III)-ligand complex can remain solubilized and stable even at temperatures of 40 to 95 DEG C.

Description

Background Art

[0001] Nitric oxide (NO) is a gaseous signaling molecule that is an important biological messenger in vertebrates. In vivo, NO is endogenously biosynthesized from L-arginine, oxygen, and NADPH by a variety of nitric oxide synthase (NOS) enzymes. Nitric oxide may also be generated by the reduction of inorganic nitrates. In the presence of iron, nitric oxide binds to the heme region of the enzyme leading to activation.

[0002] Nitric oxide plays an important role in many physiological and pathological processes. Nitric oxide can diffuse through cell membranes without an intermediate transport mechanism and can therefore signal to neighboring cells or tissues in an efficient and rapid manner. For example, nitric oxide produced by vascular endothelial cells can signal the surrounding vascular smooth muscle to relax, resulting in vasodilation and increased blood flow. Nitric oxide may also participate in electron transport and redox reactions in biochemical events in human cells. NO may cause various physiological effects, such as endothelium-dependent vasodilation, by activating guanylate cyclase.

[0003] Inhaled nitric oxide can improve the body's oxidative capacity and reduce the need for high-risk extracorporeal cardiopulmonary support in critically ill patients. Controlled administration of appropriate amounts of inhaled nitric oxide can reduce pulmonary hypertension and improve oxygenation. Inhaled nitric oxide has been approved as a drug by the U.S. Food and Drug Administration for the treatment of persistent pulmonary hypertension of the newborn. Inhaled nitric oxide therapy has also been used in various diseases or clinical medicine fields, such as neonatal respiratory diseases, intensive care medicine, cardiothoracic surgery, acute respiratory distress, and anesthesiology. Summary of the invention

[0004] The present disclosure provides an apparatus and method for producing nitric oxide gas, which can be performed on demand. The method can include electrolyzing an iron (III)-ligand complex in a medium containing nitrite ions. The medium preferably has a pH of 5 to 9, wherein the iron (III)-ligand complex can remain dissolved and stable even at a temperature of 40-95°C.

[0005] According to one embodiment of the present disclosure, there is provided a device for generating nitric oxide, comprising: a working electrode and a reference or counter electrode, a medium in contact with the working electrode and the reference or counter electrode, wherein the medium contains nitrite ions, an iron (III)-ligand complex in contact with the working electrode, and an outlet for releasing the nitric oxide generated from the medium.

[0006] According to some embodiments, the ligand is an oxygen-containing or nitrogen-containing iron complexing agent. In some embodiments, the iron (III)-ligand complex is stable at 40-95° C. for at least 1 hour.

[0007] In some embodiments, the ligand is selected from Table A.

[0008] In some embodiments, the ligand is selected from the group consisting of nitrilotriacetic acid (NTA) or a salt thereof, 8-hydroxyquinoline-5-sulfonic acid (HQSA) or a salt thereof, citric acid (CA) or a salt thereof, and N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) or a salt thereof.

[0009] In some embodiments, the iron (III)-ligand complex is selected from the group consisting of:

[0010]

[0011] and

[0012] In some embodiments, the iron (III)-ligand complex is present at a concentration of 1.0 to 70.0 mM.

[0013] In some embodiments, the pH of the medium is from 5 to 9, or preferably from 5.5 to 8.5, 6.5 to 8.2, or 6.8 to 8.0.

[0014] In some embodiments, the medium comprises a buffer.

[0015] In some embodiments, the buffer comprises a reagent selected from the group consisting of: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof, 3-(N-morpholino)propanesulfonic acid (MOPS) or a salt thereof, tris(hydroxymethyl)aminomethane (Tris) or a salt thereof, barbiturate or a salt thereof, phosphoric acid or a salt thereof, boric acid or a salt thereof, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) or a salt thereof, N-tris(hydroxymethyl)methylglycine (Tricine) or a salt thereof, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) or a salt thereof. its salts, N,N-bis(2-hydroxyethyl)glycine (Bicine) or its salts, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) or its salts, dimethylarsonic acid or its salts, citric acid or its salts, 2-(N-morpholino)ethanesulfonic acid (MES) or its salts, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) or its salts, 2,2-bis(hydroxyethyl)-(iminotris)-(hydroxymethyl)-methane (Bis-Tris) or its salts, glycine-hydrochloric acid, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl), and combinations thereof.

[0016] In some embodiments, the concentration of the buffer is 0.05 to 1.5M.

[0017] In some embodiments, the nitrite ions are provided as inorganic or organic nitrite.

[0018] In some embodiments, the nitrite ions are provided as NaNO2.

[0019] In some embodiments, the nitrite ion is present at a concentration of 0.05 to 7.0 mM.

[0020] In some embodiments, an inlet is also included for delivering a carrier gas into the medium.

[0021] In another embodiment, a method for producing nitric oxide is also provided, comprising electrolyzing an iron (III)-ligand complex in a medium containing nitrite ions to produce nitric oxide, wherein: the pH of the medium is 5 to 9, and the iron (III)-ligand complex is soluble and stable in the medium at a temperature of 40-95°C for at least 1 hour.

[0022] In another embodiment, the iron(III)-ligand complex is soluble and stable in the medium when the pH of the medium is from 5.5 to 8.5, from 6.5 to 8.2, or from 6.8 to 8.0.

[0023] According to some embodiments, the ligand is an oxygen-containing or nitrogen-containing iron complexing agent.

[0024] In some embodiments, the ligand is selected from Table A.

[0025] In some embodiments, the ligand is selected from the group consisting of nitrilotriacetic acid (NTA) or a salt thereof, 8-hydroxyquinoline-5-sulfonic acid (HQSA) or a salt thereof, citric acid (CA) or a salt thereof, and N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) or a salt thereof.

[0026] In some embodiments, the iron (III)-ligand complex is selected from the group consisting of:

[0027]

[0028] and

[0029] In some embodiments, the iron (III)-ligand complex is present at a concentration of 1.0 to 70.0 mM.

[0030] In some embodiments, the medium comprises a buffer.

[0031] In some embodiments, the buffer comprises a reagent selected from the group consisting of: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof, 3-(N-morpholino)propanesulfonic acid (MOPS) or a salt thereof, tris(hydroxymethyl)aminomethane (Tris) or a salt thereof, barbiturate or a salt thereof, phosphoric acid or a salt thereof, boric acid or a salt thereof, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) or a salt thereof, N-tris(hydroxymethyl)methylglycine (Tricine) or a salt thereof, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) or a salt thereof. its salts, N,N-bis(2-hydroxyethyl)glycine (Bicine) or its salts, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) or its salts, dimethylarsonic acid or its salts, citric acid or its salts, 2-(N-morpholino)ethanesulfonic acid (MES) or its salts, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) or its salts, 2,2-bis(hydroxyethyl)-(iminotris)-(hydroxymethyl)-methane (Bis-Tris) or its salts, glycine-hydrochloric acid, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl), and combinations thereof.

[0032] In some embodiments, the concentration of the buffer is 0.05 to 1.5M.

[0033] In some embodiments, the nitrite ions are provided as inorganic or organic nitrite.

[0034] In some embodiments, the nitrite ions are provided as NaNO2.

[0035] In some embodiments, the nitrite ion is present at a concentration of 0.05 to 7.0 mM.

[0036] In some embodiments, the method further comprises delivering a carrier gas into the medium to carry the nitric oxide generated from the medium out of the medium.

[0037] In some embodiments, the method further comprises regulating the production of nitric oxide by regulating the potential or current applied to the electrolysis.

[0038] In some embodiments, the method further comprises regulating the generation of nitric oxide by adjusting the flow rate of the carrier gas.

[0039] In some embodiments, the method further comprises regulating the production of nitric oxide by regulating the contact area between the iron (III)-ligand complex and a working electrode used to apply electrolysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1AShown are nitric oxide (NO) concentrations measured at different current levels (40 mA, 20 mA, and 10 mA) using the complex Fe-trisodium nitrilotriacetate (NTA). Figure 1B A correlation plot between current and NO concentration is shown.

[0041] Figure 2 Shown are nitric oxide (NO) concentrations measured at different current levels (40 mA and 20 mA) using the complex Fe-8-hydroxyquinoline-5-sulfonic acid (HQSA).

[0042] Figure 3A Shown are the nitric oxide (NO) concentrations measured at different current levels (40 mA, 20 mA and 10 mA) using the complex Fe-citrate (CA). Figure 3B A correlation plot between current and NO concentration is shown.

[0043] Figure 4 Shown are nitric oxide (NO) concentrations measured at different current levels (40 mA, 20 mA, and 10 mA) using the complex Fe-N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA).

[0044] Figure 5 Shown are the nitric oxide (NO) concentrations measured at different current levels (10 mA and 20 mA) using the complex Fe-porphyrin.

[0045] Figure 6 Shown is the nitric oxide (NO) concentration measured using the complex Cu-citrate (CA) at a current of 40 mA.

[0046] Figure 7 Apparatus for producing nitric oxide according to various examples are shown.

[0047] Figure 8 The correlation between the nitric oxide (NO) generation rate and the NaNO2 concentration is shown.

[0048] Fig. 9 It is shown that when the nitrite ion concentration is lower than 5M, a higher nitrite ion concentration decreases the melting temperature (or solidification temperature) of the electrolyte; whereas when the nitrite ion concentration is higher than 5M, a higher nitrite ion concentration results in an increased melting temperature of the electrolyte.

[0049] Fig.10 It is shown that the system has similar nitric oxide (NO) generation rates using 5 M NaNO2 at different operating temperatures.

[0050] Fig.11Shown is the correlation between the nitric oxide (NO) generation rate and the NaNO2 concentration when HEDTA is used as a ligand.

[0051] It will be appreciated that some or all of the figures are schematic representations for illustrative purposes. DETAILED DESCRIPTION

[0052] definition

[0053] The following description sets forth exemplary embodiments of the present technology. However, it should be appreciated that such description is not intended to limit the scope of the present disclosure, but rather is a description of exemplary embodiments.

[0054] As used in this specification, the following words, phrases and symbols are generally intended to have the meanings set forth below, unless the context in which they are used indicates otherwise.

[0055] A dash ("-") that is not between two letters or symbols is used to indicate the point of attachment of a substituent. For example, -C(O)NH2 is attached through the carbon atom. Dashes at the beginning or end of a chemical group are for convenience; a chemical group may be represented with or without one or more dashes without losing its usual meaning. A wavy line drawn through a line in a structure indicates the point of attachment of a group. Unless chemically or structurally required, the order in which chemical groups are written or named does not indicate or imply any directionality.

[0056] References to "about" values ​​or parameters herein include (and describe) embodiments for the value or parameter itself. In certain embodiments, the term "about" includes an indication of ±10%. In other embodiments, the term "about" includes an indication of ±5%. In certain other embodiments, the term "about" includes an indication of ±1%. In addition, the term "about X" includes a description of "X". In addition, unless the context clearly states otherwise, nouns that are not limited by quantifiers include singular and / or plural references. Thus, for example, references to "compounds" include a variety of such compounds, and references to "assays" include references to one or more assays known to those skilled in the art and their equivalents.

[0057] "Alkyl" refers to an unbranched or branched saturated hydrocarbon chain. As used herein, an alkyl group has 1 to 20 carbon atoms (i.e., C 1-20 Alkyl), 1 to 8 carbon atoms (i.e. C 1-8 Alkyl), 1 to 6 carbon atoms (i.e. C 1-6 Alkyl) or 1 to 4 carbon atoms (i.e. 1-4Alkyl). Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. When an alkyl residue having a particular number of carbon atoms is named by chemical name or identified by molecular formula, all positional isomers having that number of carbon atoms are contemplated; thus, for example, "butyl" includes n-butyl (i.e., -(CH2)3CH3), sec-butyl (i.e., -CH(CH3)CH2CH3), isobutyl (i.e., -CH2CH(CH3)2), and tert-butyl (i.e., -C(CH3)3); and "propyl" includes n-propyl (i.e., -(CH2)2CH3) and isopropyl (i.e., -CH(CH3)2).

[0058] “Alkoxy” refers to the group “alkyl-O—.” Examples of alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2-dimethylbutoxy.

[0059] "Haloalkoxy" refers to an alkoxy group as defined above wherein one or more hydrogen atoms are replaced by a halogen.

[0060] "Acyl" refers to a group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, heteroalkyl, or heteroaryl; each of which may be optionally substituted as defined herein. Examples of acyl include formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethyl-carbonyl, and benzoyl.

[0061] "Amino" refers to the group -NR y R z , where R y and R z are independently selected from the group consisting of hydrogen, alkyl, haloalkyl, aryl or heteroaryl; each of which may be optionally substituted.

[0062] "Aryl" refers to an aromatic carbocyclic group having a single ring (e.g., monocyclic) or multiple rings (e.g., bicyclic or tricyclic), including fused systems. As used herein, an aryl group has 6 to 20 ring carbon atoms (i.e., C 6-20 aryl), 6 to 12 ring carbon atoms (i.e., C 6-12 aryl) or 6 to 10 ring carbon atoms (i.e. C 6-10 The term "aryl" refers to a group consisting of a phenyl group, a naphthyl group, a fluorenyl group, and an anthracenyl group. The term "aryl" refers to a group consisting of a phenyl group, a naphthyl group, a fluorenyl group, and an anthracenyl group. However, an aryl group does not in any way encompass or overlap with a heteroaryl group as defined below. If one or more aryl groups are fused to a heteroaryl group, the resulting ring system is a heteroaryl group. If one or more aryl groups are fused to a heterocyclyl group, the resulting ring system is a heterocyclyl group.

[0063] "Carboxy" refers to -C(O)OH.

[0064] "Carboxylate" refers to both -OC(O)R and -C(O)OR, where R is hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, heteroalkyl, or heteroaryl; each of which may be optionally substituted as defined herein.

[0065] "Cycloalkyl" refers to a saturated or partially unsaturated cyclic alkyl group, having a single ring or multiple rings, including fused rings, bridged rings, and spiro ring systems. The term "cycloalkyl" includes cycloalkenyl groups (i.e., cyclic groups having at least one double bond). As used herein, a cycloalkyl group has 3 to 20 ring carbon atoms (i.e., C 3-20 Cycloalkyl), 3 to 12 ring carbon atoms (i.e., C 3-12 Cycloalkyl), 3 to 10 ring carbon atoms (i.e. C 3-10 Cycloalkyl), 3 to 8 ring carbon atoms (i.e. C 3-8 Cycloalkyl) or 3 to 6 ring carbon atoms (i.e. C 3-6 Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0066] "Imino" refers to the group -C(NR)R, where each R is alkyl, cycloalkyl, heterocyclyl, aryl, heteroalkyl, or heteroaryl; each group being optionally substituted as defined herein.

[0067] "Halogen" or "halo" includes fluorine, chlorine, bromine and iodine. "Haloalkyl" refers to an unbranched or branched alkyl group as defined above, in which one or more hydrogen atoms are replaced by halogen. For example, when a residue is replaced by more than one halogen, a prefix corresponding to the number of halogen moieties connected can be used to refer to it. Dihaloalkyl and trihaloalkyl refer to alkyl substituted by two ("two") or three ("three") halogen groups, which can be but are not necessarily identical halogens. Examples of haloalkyl include difluoromethyl (-CHF2) and trifluoromethyl (-CF3).

[0068] "Heteroalkyl" refers to an alkyl group in which one or more carbon atoms (and any associated hydrogen atoms) are each independently substituted by the same or different heteroatom groups. The term "heteroalkyl" includes an unbranched or branched saturated chain with carbon and heteroatoms. For example, 1, 2 or 3 carbon atoms can be independently substituted by the same or different heteroatom groups. Heteroatom groups include but are not limited to-NR-,-O-,-S-,-S(O)-,-S(O)2-, etc., wherein R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl or heterocyclic radical, wherein each group can be optionally substituted. Examples of heteroalkyl groups include-OCH3,-CH2OCH3,-SCH3,-CH2SCH3,-NRCH3 and-CH2NRCH3, wherein R is hydrogen, alkyl, aryl, aralkyl, heteroalkyl or heteroaryl, wherein each group can be optionally substituted. As used herein, a heteroalkyl group includes 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 4 carbon atoms; and 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom.

[0069] "Heteroaryl" refers to an aromatic group having a single ring, multiple rings, or multiple fused rings, wherein one or more ring heteroatoms are independently selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl group includes 1 to 20 ring carbon atoms (i.e., C 1-20 heteroaryl), 3 to 12 ring carbon atoms (i.e., C 3-12 heteroaryl) or 3 to 8 ring carbon atoms (i.e., C 3-8 Heteroaryl);And 1 to 5 heteroatoms, 1 to 4 heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms or 1 ring heteroatom, heteroatoms are independently selected from nitrogen, oxygen and sulfur. Examples of heteroaryl groups include pyrimidinyl, purinyl, pyridyl, pyridazinyl, benzothiazolyl and pyrazolyl. Examples of fused heteroaromatic rings include, but are not limited to, benzo[d]thiazolyl, quinolyl, isoquinolyl, benzo[b]thienyl, indazolyl, benzo[d]imidazolyl, pyrazolo[1,5-a]pyridyl and imidazo[1,5-a]pyridyl, wherein the heteroaryl can be combined by any ring of the fused system. Any aromatic ring having one or more fused rings and containing at least one heteroatom is considered a heteroaryl, regardless of how it is connected to the rest of the molecule (i.e., through any fused ring). Heteroaryl does not cover or overlap with the aryl defined above.

[0070] "Heterocyclic radical" refers to a saturated or unsaturated cyclic alkyl group having one or more ring heteroatoms independently selected from nitrogen, oxygen and sulfur. The term "heterocyclic radical" includes heterocyclic alkenyl groups (i.e., heterocyclic groups with at least one double bond), bridged heterocyclic groups, fused heterocyclic groups and spiral heterocyclic groups. The heterocyclic radical can be a monocyclic or polycyclic ring, wherein the polycyclic ring can be fused, bridged or spiral. Any non-aromatic ring containing at least one heteroatom is considered as a heterocyclic radical, regardless of its mode of attachment (i.e., it can be combined by carbon atoms or heteroatoms). In addition, the term heterocyclic radical is intended to encompass any non-aromatic ring containing at least one heteroatom, which ring can be fused to an aryl or heteroaryl ring, regardless of its mode of attachment to the rest of the molecule. As used herein, a heterocyclic radical has 2 to 20 ring carbon atoms (i.e., C 2-20 heterocyclic group), 2 to 12 ring carbon atoms (i.e., C 2-12 heterocyclic group), 2 to 10 ring carbon atoms (i.e., C 2-10 heterocyclyl), 2 to 8 ring carbon atoms (i.e., C 2-8 heterocyclic group), 3 to 12 ring carbon atoms (i.e., C 3-12 heterocyclic group), 3 to 8 ring carbon atoms (i.e., C 3-8 heterocyclic) or 3 to 6 ring carbon atoms (i.e., C 3-6 Heterocyclic radical);Having 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms or 1 ring heteroatom, the heteroatom is independently selected from nitrogen, sulfur or oxygen. Examples of heterocyclic radical groups include pyrrolidinyl, piperidinyl, piperazinyl, oxetanyl, dioxolanyl, azetidinyl and morpholinyl. As used herein, the term "bridged heterocyclic radical" refers to a four to ten-membered cyclic moiety, which is connected to one or more (e.g., 1 or 2) four to ten-membered cyclic moieties having at least one heteroatom at two non-adjacent atoms of a heterocyclic radical, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur. As used herein, bridged heterocyclic radicals include bicyclic and tricyclic systems. As used herein, the term "spiroheterocyclyl" refers to a ring system in which a three- to ten-membered heterocyclyl has one or more additional rings, wherein the one or more additional rings are three- to ten-membered cycloalkyl or three- to ten-membered heterocyclyl, wherein the single atoms of the one or more additional rings are also atoms of the three- to ten-membered heterocyclyl. Examples of spiroheterocyclyl rings include bicyclic and tricyclic ring systems, such as 2-oxa-7-azaspiro[3.5]nonyl, 2-oxa-6-azaspiro[3.4]octyl, and 6-oxa-1-azaspiro[3.3]heptyl. Examples of fused heterocyclyl rings include, but are not limited to, 1,2,3,4-tetrahydroisoquinolyl, 4,5,6,7-tetrahydrothieno[2,3-c]pyridinyl, indolyl, and isoindolyl, wherein the heterocyclyl may be bound by any ring of the fused system.

[0071] "Hydroxide" or "hydroxyl" refers to the radical -OH.

[0072] "Oxo" refers to the group (=O) or (O).

[0073] "Nitro" refers to the radical -NO2.

[0074] "Sulfonyl" refers to the group -S(O)2R, where R is alkyl, haloalkyl, heterocyclyl, cycloalkyl, heteroaryl or aryl. Examples of sulfonyl are methylsulfonyl, ethylsulfonyl, phenylsulfonyl and toluenesulfonyl.

[0075] Certain commonly used alternative chemical names may be used. For example, divalent groups, such as divalent "alkyl" groups, divalent "aryl" groups, etc., may also be referred to as "alkylene" groups or "alkylenyl" groups, "arylene" groups or "arylenyl" groups, respectively. In addition, unless otherwise expressly stated, when a combination of groups is referred to herein as a moiety (e.g., arylalkyl), the last mentioned group includes the atoms to which the moiety is attached to the rest of the molecule.

[0076] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. In addition, the term "optionally substituted" means that any one or more hydrogen atoms on the designated atom or group may or may not be replaced with a moiety other than hydrogen.

[0077] Some compounds exist as tautomers. Tautomers are in equilibrium with each other. For example, compounds containing amides may reach equilibrium with imidic acid tautomers. No matter which tautomer is shown, no matter how the equilibrium properties between the tautomers are, those of ordinary skill in the art understand that these compounds include both amides and imidic acid tautomers. Therefore, amide-containing compounds are understood to include their imidic acid tautomers. Similarly, imidic acid-containing compounds are understood to include their amide tautomers.

[0078] Any formula or structure given herein is also intended to represent unlabeled forms of the compound as well as isotopically labeled forms. An isotopically labeled compound has a structure as shown by a formula given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to, 2 H (deuterium, D), 3 H (tritium), 11 C. 13 C. 14 C. 15 N. 18 F. 31 P. 32 P.35 S. 36 Cl and 125 I. Various isotopically labeled compounds of the present disclosure, for example, compounds into which a radioactive isotope (e.g. 3 H. 13 C and 14 Such isotopically labeled compounds are useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distribution analysis or radiotherapy of patients.

[0079] The present disclosure also includes "deuterated analogs" of compounds of Formula I, wherein 1 to n hydrogens attached to the carbon atom are replaced by deuterium, where n is the number of hydrogens in the molecule. Such compounds exhibit increased resistance to metabolism and can therefore be used to increase the half-life of any compound of Formula I when administered to mammals, particularly humans. See, e.g., Foster, "Deuterium Isotope Effects in Studies of Drug Metabolism," Trends Pharmacol. Sci. 5(12): 524-527 (1984). Such compounds are synthesized by methods well known in the art, for example by using starting materials in which one or more hydrogens have been replaced by deuterium.

[0080] Deuterium-labeled or substituted therapeutic compounds of the present disclosure may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes (e.g., deuterium) may provide certain therapeutic advantages due to greater metabolic stability (e.g., increased in vivo half-life), reduced dosage requirements and / or improved therapeutic index. 18 F-labeled compounds may be suitable for PET or SPECT studies. Isotope-labeled compounds and prodrugs thereof disclosed herein can generally be prepared by performing the procedures disclosed in the schemes or the examples and preparations described below by replacing non-isotope-labeled reagents with readily available isotope-labeled reagents. It should be understood that in this context, deuterium is considered a substituent in the compounds of Formula I.

[0081] The concentration of such heavier isotopes, particularly deuterium, can be defined by an isotopic enrichment factor. In the compounds of the present disclosure, any atom not explicitly designated as a particular isotope represents any stable isotope of that atom. Unless otherwise indicated, when a position is explicitly designated as "H" or "hydrogen", that position is understood to have hydrogen in its natural abundance isotopic composition. Therefore, in the compounds of the present disclosure, any atom explicitly designated as deuterium (D) represents deuterium.

[0082] In many cases, the compounds of the present disclosure are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0083] The term "substituted" refers to that any one or more hydrogen atoms on a specified atom or group are replaced by one or more non-hydrogen substituents, but the normal valence of the specified atom must not be exceeded. The one or more substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amide, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, guanidino, halogen, haloalkyl, haloalkoxy, assorted alkyl, heteroaryl, heterocyclic radical, hydroxyl, hydrazine, imino, oxo, nitro, alkylsulfinyl, sulfonic acid, alkylsulfonyl, thiocyanate, mercaptan, thioketone or its combination. The polymer or similar indefinite structure obtained by defining the substituent with unlimited additional other substituents (for example, the aryl substituted has the alkyl substituted, and the alkyl substituted itself is replaced by the aryl substituted, and the aryl substituted is further replaced by the assorted alkyl substituted, etc.) is not intended to be included in this article. Unless otherwise indicated, the maximum number of continuous substitutions in compounds described herein is three. For example, the continuous substitution of a substituted aryl group by two other substituted aryl groups is limited to an aryl substituted by ((substituted aryl) substituted aryl). Similarly, the above definition is not intended to include unallowed substitution patterns (e.g., methyl substituted by 5 fluorines or heteroaryl groups with two adjacent oxygen ring atoms). Such unallowed substitution patterns are well known to those skilled in the art. When used to modify a chemical group, the term "substituted" can describe other chemical groups defined herein. Unless otherwise specified, when a group is described as optionally substituted, any substituent of the group itself is unsubstituted. For example, in some embodiments, the term "substituted alkyl" refers to an alkyl group with one or more substituents, the substituents including hydroxyl, halogen, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl. In other embodiments, one or more substituents may be further substituted by halogen, alkyl, haloalkyl, hydroxyl, alkoxy, cycloalkyl, heterocyclic, aryl or heteroaryl, wherein each substituent is substituted. In other embodiments, the substituents may be further substituted with halogen, alkyl, haloalkyl, alkoxy, hydroxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each substituent is unsubstituted.

[0084] Production of nitric oxide (NO)

[0085] Methods for generating nitric oxide from electrochemical reactions have been developed. For example, in PCT application WO2022127902A1, the applicant discloses methods and apparatus for implementing a copper (Cu) (II) based nitric oxide production mechanism. Complexed copper (II) ions (i.e., in a Cu (II)-ligand complex) are reduced to copper (I) by electrolysis:

[0086] Cu 2+ +e - →Cu +

[0087] It can then react with nitrite ions in the medium to produce nitric oxide gas:

[0088] Cu + +NO2 - +2H + →NO+Cu 2+ +H2O.

[0089] An example of a Cu(II)-ligand complex is Cu(II)-1,4,7-trimethyl-1,4,7-triazacyclononane (Me3TACN). A significant limitation of Cu(II)-based systems is that all ligands commonly used to prepare Cu(II)-ligand complexes are only stable at low temperatures (e.g., 2-8°C). At temperatures of 35°C or higher, these Cu(II)-ligand complexes have a strong tendency to degrade. Moreover, these ligands are very expensive.

[0090] It is thought that developing nitric oxide-generating chemistries based on other metals, such as iron or cobalt, may encounter significant obstacles. The hydrated pK1 of copper(II) ions is known to be 8.0; the pK1 of iron(II) ions is 6.74, while the pK1 of iron(III) ions is 2.83 and the pK2 is 4.59. However, at pH 5.5 or lower, nitrite solutions are very resistant to H + It is highly reactive, which is detrimental to the electrolysis process. Moreover, iron(III) will precipitate at such low pH. As can be seen from the above, the development of stable electrolytes based on iron(III) ions is a major challenge.

[0091] The inventors unexpectedly discovered through trial and error that certain nitrogen- and oxygen-containing ligands can be successfully complexed with iron (III) ions, and the resulting complexes remain soluble and stable at near-neutral pH (e.g., 5 to 9) and elevated temperatures (e.g., 40-95° C.). Equally important, many of these ligands are much cheaper than Me3TACN.

[0092] Another surprising finding is that all the iron (III)-ligand-based systems tested exhibited much higher (30-90%) Faradaic efficiencies than the copper (II)-ligand-based systems (see Comparative Example 3, 9.34%). Thus, such a finding enables the technician to generate nitric oxide under more convenient conditions (e.g., weakly acidic or alkaline or neutral pH compared to highly acidic pH; room temperature or higher compared to low temperature) with greatly improved efficiency and much lower cost.

[0093] Therefore, according to one embodiment of the present disclosure, a method for generating nitric oxide (NO) is provided, the method comprising electrolyzing an iron (III)-ligand complex in a medium containing nitrite ions. NO can be generated or delivered at a predetermined concentration and / or flow rate. For example, NO can be generated at a clinically relevant concentration and / or flow rate for inhaled NO therapy. The concentration and / or flow rate of NO in the product gas can be adjusted. The concentration range of NO in the product gas can be, for example, from about 0 to about 20,000 ppm.

[0094] A. Ligands and Iron(III)-ligand complexes

[0095] In some embodiments, the ligand forming the iron (III)-ligand complex is an iron complexing agent. The term "iron complexing agent" refers to a chemical substance that can bind to an iron ion through its single or multiple sites. These sites have lone pairs of electrons that can be donated to the d orbitals of the metal ion, forming coordination bonds. This produces a coordination compound. The iron complexing agent can surround the iron ion or can act as a bridge between two iron ions. The iron complexing agent can have one, two or more binding sites with the iron ion. In some embodiments, the iron complexing agent is an iron (III) complexing agent.

[0096] In some embodiments, the iron complexing agent contains one or more nitrogen atoms. In some embodiments, the iron complexing agent contains one or more oxygen atoms. In some embodiments, the iron complexing agent contains both nitrogen atoms and oxygen atoms.

[0097] In some embodiments, the iron (III)-ligand complex of the present disclosure is soluble in a medium having a pH of 5 to 9. In some embodiments, the solubility in the medium is at least 0.1 mM. In some embodiments, the solubility in the medium is at least 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, or 50 mM. In some embodiments, when the pH is 5.5 to 9, or 5.5 to 8.5, 6.5 to 8.2 or 6.8 to 8.0, the solubility in the medium is at least 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM or 50 mM, but is not limited thereto.

[0098] In some embodiments, the iron (III)-ligand complexes of the present disclosure are stable at temperatures of 30° C. or more and / or below 95° C., 90° C., 85° C., or 80° C. In some embodiments, the iron (III)-ligand complexes of the present disclosure are stable at temperatures of 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., or 75° C. or more.

[0099] In some embodiments, the iron (III)-ligand complex is stable in a medium under certain conditions, meaning that the complex does not degrade and / or form a precipitate in the medium for at least 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 1 day, 2 days, 1 week, 2 weeks, 1 month, 2 months, or 6 months. In some embodiments, the conditions include pH levels and / or temperatures as described above. For example, the pH can be 5 to 9, 5.5 to 8.5, 6.5 to 8.2, or 6.8 to 8.0, but is not limited thereto. In some embodiments, the temperature is 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, or 75°C or higher.

[0100] It should be understood that iron (II) -ligand complexes can be prepared and used in the present technology, and then can be oxidized to iron (III) -ligand complexes. In some embodiments, oxidation does not require additional reactants because oxygen in the air or medium can oxidize the iron (II) -ligand complex.

[0101] Non-limiting examples of iron complexing agents (ligands) and corresponding iron-ligand complexes are provided in Table A below.

[0102] Table A. Exemplary ligands and corresponding complexes

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] In a specific embodiment, the iron complexing agent (ligand) is nitrilotriacetic acid (NTA) or a salt thereof. In another embodiment, the iron complexing agent (ligand) is 8-hydroxyquinoline-5-sulfonic acid (HQSA) or a salt thereof. In another embodiment, the iron complexing agent (ligand) is citric acid (CA) or a salt thereof. In another embodiment, the iron complexing agent (ligand) is N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) or a salt thereof. Examples thereof corresponding to iron (III)-ligand complexes are shown in the table above.

[0111] In some embodiments, the iron (III)-ligand complex is provided in a medium in which electrolysis is performed. In some embodiments, the iron (III)-ligand complex is present in the medium at a concentration of 0.1 to 100.0 mM. In some embodiments, the iron (III)-ligand complex is present in the medium at a concentration of 0.1 to 50.0 mM. In some embodiments, the iron (III)-ligand complex is present in the medium at a concentration of 0.1 to 100.0 mM, 0.5 to 100.0 mM, 0.5 to 90.0 mM, 0.5 to 80.0 mM, 0.5 to 75.0 mM, 0.5 to 70.0 mM, 0.5 to 65.0 mM, 0.5 to 60.0 mM, 0.5 to 55.0 mM, 0.5 to 50.0 mM, 1.0 to 100.0 mM, 1.0 to 90.0 mM, 1.0 to 80.0 m In some embodiments, the iron (III)-ligand complex is present at a concentration of at least 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, or 40 mM. In some embodiments, the iron (III)-ligand complex is present at a concentration of no greater than 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, or 10 mM.

[0112] In some embodiments, the iron (III)-ligand complex is attached or otherwise immobilized to the working electrode while in contact with the medium. For example, immobilization may require that the iron (III)-ligand complex is covalently attached to the surface of the working electrode. In another example, the iron (III)-ligand complex can be physically adsorbed to the working electrode, or doped or covalently attached to a polymer, film, or hydrogel deposited on the surface of the working electrode.

[0113] B. Electrolytes

[0114] The medium comprising nitrite ions is used as an electrolyte in the electrochemical process. In some embodiments, the medium comprises at least a suitable buffer (or buffering agent) to provide and / or maintain a desired pH.

[0115] In some embodiments, the buffer can be any commonly used buffer, as long as it does not interfere with the stability or solubility of the iron (III)-ligand complex. Many such useful buffers are known, and non-limiting examples include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof, 3-(N-morpholino)propanesulfonic acid (MOPS) or a salt thereof, tris(hydroxymethyl)aminomethane (Tris) or a salt thereof, barbital or a salt thereof, phosphoric acid or a salt thereof, boric acid or a salt thereof, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) or a salt thereof, N-tris(hydroxymethyl)methylglycine (Tricine) or a salt thereof, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) or a salt thereof. or its salts, N,N-bis(2-hydroxyethyl)glycine (Bicine) or its salts, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) or its salts, dimethylarsonic acid or its salts, citric acid or its salts, 2-(N-morpholino)ethanesulfonic acid (MES) or its salts, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) or its salts, 2,2-bis(hydroxyethyl)-(iminotris)-(hydroxymethyl)-methane (Bis-Tris) or its salts, glycine-hydrochloric acid, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl).

[0116] The buffer may have a concentration suitable for providing a desired pH. In one embodiment, the concentration is 0.01 to 5.0 M. In one embodiment, the concentration is 0.01 to 4.0 M, 0.02 to 3.0 M, 0.02 to 2.5 M, 0.02 to 2.0 M, 0.05 to 2.0 M, 0.05 to 1.0 M, 0.05 to 1.5 M, 0.05 to 1.2 M, 0.1 to 0.9 M, 0.2 to 0.8 M, or 0.3 to 0.7 M, but is not limited thereto.

[0117] In some embodiments, the medium includes a nitrite source, which can be an inorganic nitrate or an organic nitrite. The inorganic nitrate can be NO2 - Examples include, but are not limited to, nitrites of Li, Na, K, Rb, Ca, Mg, Al, and Fe. In one embodiment, the nitrite is sodium nitrite (NaNO2). Examples of organic nitrites include R4N + NO2 - 、RNH3 + NO2 -, nicleosamide, nifedipine, flutamine, isobutyl nitrate, isopentyl nitrite, 1,3-propane nitrite, 1,5-pentane nitrite, 1,7-heptane nitrite and cyclohexylmethyl nitrite, but are not limited thereto.

[0118] The nitrite ions may be present in the medium at a concentration of at least 0.01 M. In some embodiments, the concentration is 0.01 to 20.0 M. In some embodiments, the concentration is 0.02 to 10.0 M. In some embodiments, the concentration is 0.05 to 7.0 M. In some embodiments, the concentration is 0.05 to 5.0 M. In some embodiments, the concentration is 0.1 to 4.0 M, 0.2 M to 2 M, 0.5 M to 1.0 M, 0.5 to 7.0 M, 0.5 to 6.0 M, 0.5 to 5.0 M, 0.5 to 4.0 M, 0.5 M to 3 M, 0.5 M to 2.0 M, or 0.5 M to 1.5 M, but are not limited thereto.

[0119] In some embodiments, the nitric oxide produced can be taken away with a carrier gas. The carrier gas may include any suitable gas, such as air, nitrogen, helium, argon, and oxygen. In some embodiments, the carrier gas includes nitrogen. In some embodiments, the nitrogen concentration in the carrier gas is about or higher than 99.0% by volume. For example, the nitrogen concentration in the carrier gas may be about or higher than 99.10%, 99.20%, 99.30%, 99.40%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 99.95%, 99.98%, or 99.99% by volume. In some embodiments, the carrier gas comprises oxygen. For example, the oxygen concentration in the carrier gas may be less than about 1%, 0.5%, or 0.1%.

[0120] In some embodiments, a carrier gas may be used to sweep across the surface of the working electrode. Sweeping across the surface of the electrode may increase the Faradaic efficiency and / or the reaction rate of the electrochemical reaction at and / or near the electrode surface and / or may increase the nitric oxide concentration of the product gas.

[0121] C. Nitric Oxide Generation and Exemplary Apparatus

[0122] As disclosed herein, nitric oxide (NO) can be generated or delivered by the present technology at a predetermined concentration and / or flow rate. For example, NO can be generated at a clinically relevant concentration and / or flow rate for inhaled NO therapy. In addition, the concentration and / or flow rate of NO in the product gas can be adjusted. The concentration range of NO in the product gas can be, for example, from about 0 to about 20,000 ppm.

[0123] The dimensionless unit "ppm" used in this disclosure to describe gas concentration refers to parts per million by volume and can be converted to other concentration units, such as parts per million by mole or milligrams per liter (mg / L). The dimensionless unit "%" or "volume %" used in this disclosure to describe gas concentration refers to volume percentage and can be converted to other concentration units, such as weight percentage or molar concentration.

[0124] In some embodiments, the present technology can allow NO generation in a session that can include at least one operating period. During the operating period, the NO concentration and / or flow rate in the product gas can reach and / or remain at a steady state. The operating period can, for example, last up to 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, or 60 hours or more.

[0125] Also provided are exemplary devices and apparatus that can be used to implement the present NO generation technology. Figure 7 As shown, the device may include a carrier gas production compartment 710, a reaction compartment 720, a purification compartment 730 and / or an output compartment 740. The carrier gas production compartment 710 produces or generates a carrier gas (e.g., nitrogen (N2)), which removes the generated nitric oxide from the reaction compartment 720. The reaction compartment 720 includes an electrolytic cell (e.g., a first electrode 722, a second electrode 724 connected to a power source 726 and activated by it), a reactant, and a micro-release control reactant. The reaction compartment 720 can store an iron-based ligand complex. As previously described, when an electric current or voltage is applied to the first electrode 722, an electrochemical reaction occurs, and the first electrode 722 can be used as a working electrode. The second electrode 724 can be a reference electrode, a counter electrode, or a combination thereof.

[0126] The purification compartment 730 can filter and absorb pollutants from the purged generated nitric oxide. The pollutants to be removed may include salt spray and / or water vapor. The output compartment 740 may include a pressure tank that stores nitric oxide and other nitrogen gases. At the output of the pressure tank, a NO2 conversion filter can remove NO2 that may have been oxidized from the nitric oxide. In some instances, oxygen can be added and mixed with NO before the product gas is supplied to the patient.

[0127] In some embodiments, the device may include one or more NO sensors (not shown) to detect the NO concentration in the product gas. The NO sensor may be arranged in any suitable position. For example, the NO sensor may be arranged to contact the product gas in the output compartment 740. In some embodiments, the NO sensor is disposed in or near the purge compartment 730.

[0128] In some embodiments, the catalyst can be immobilized on the surface of an electrode (e.g., working electrode 722). In some embodiments, the catalyst comprises one or more compounds selected from the group consisting of cystine, cysteine, methionine, thiophene, and derivatives thereof. For example, one or more catalysts can be covalently attached to, adsorbed to, or doped in or covalently attached to a material (e.g., a polymer, a film, or a hydrogel) deposited on an electrode.

[0129] In some embodiments, the working electrode 722 and / or the reference / counter electrode 724 can have any suitable shape including one or more surfaces. For example, the working electrode 722 can include a plate, a sheet, or a mesh. In some embodiments, when a cathode voltage is applied to the working electrode 722, or when a cathode current is applied to the working electrode 722, NO is electrochemically generated by one or more electrochemical reactions occurring at and / or near one or more surfaces of the working electrode 722.

[0130] The power supply 726 may include one or more suitable power supply devices or circuits that allow voltage or current to be applied to the electrode, such as an electrical socket, a power circuit, a DC power supply, an AC power supply, a generator, or an energy storage device. The energy storage device may include, for example, one or more batteries or fuel cells. In some embodiments, the power supply 726 includes one or more circuits for controlling or regulating the voltage or current applied to the electrode. In some embodiments, one or more circuits may include a potentiostat to control or regulate the voltage applied to the electrode. In some embodiments, one or more circuits may include a galvanostat to control or regulate the current through the electrode.

[0131] The electrode can be made of one or more materials, such as a conductive material. The conductive material can be a metal or a non-metallic material. Non-limiting examples of conductive materials include platinum, palladium, gold, copper, brass, silver, carbon, glassy carbon, boron-doped diamond (BDD), graphite, stainless steel, titanium, iridium, ruthenium, and one or more alloys thereof, such as ruthenium-iridium alloys.

[0132] In some embodiments, the electrode may include a substrate material. In some embodiments, the conductive material is coated on the substrate material. The coating may be performed using any suitable coating method, such as electroplating, physical vapor deposition (PVD), chemical vapor deposition (CVD), or plasma enhanced chemical vapor deposition (PECVD).

[0133] Controlling the magnitude of the voltage or current applied to the working electrode allows controlling the ratio of the reduced form of the iron (III)-ligand complex to its oxidized form at and / or near the electrode surface. This therefore allows controlling the amount and / or rate of NO produced at a given concentration of nitrite ions and iron (III)-ligand complex in the medium.

[0134] In some embodiments, the voltage applied to the electrode is a DC voltage. In some embodiments, the voltage ranges from about 0.1V to about 5.0V, such as about 0.1V to about 4.0V, about 0.2V to about 3.0V, about 0.4V to about 2.0V, or about 0.5V to about 1.5V, but is not limited thereto.

[0135] In some embodiments, the current applied to the electrode is a DC current. In some embodiments, the current ranges from about 0 mA to about 600 mA, such as about 0 mA to about 10 mA, about 10 mA to about 50 mA, about 50 mA to about 100 mA, about 100 mA to about 200 mA, about 200 mA to about 300 mA, about 300 mA to about 400 mA, about 400 mA to about 500 mA, or about 500 mA to about 600 mA.

[0136] In some embodiments, the medium is maintained at or near the reaction temperature or within a temperature range. The electrochemical reaction in the reaction compartment 720 may have the highest, desired or optimized reaction rate and / or Faraday efficiency at or near the reaction temperature or within a temperature range. The reaction temperature or temperature range may be determined based on one or more conditions, such as buffer and / or catalyst components and concentrations in the reaction medium. In some embodiments, the reaction temperature or temperature range may be about 5°C to about 80°C, about 10°C to about 80°C, about 15°C to about 80°C, about 20°C to about 80°C, about 30°C to about 80°C, about 40°C to about 80°C, about 50°C to about 80°C, about 55°C to about 80°C, about 60°C to about 80°C, or a combination thereof.

[0137] Therefore, according to one embodiment of the present disclosure, a device for generating nitric oxide is provided, which includes a working electrode and a reference or counter electrode, a medium in contact with the working electrode and the reference or counter electrode, wherein the medium contains nitrite ions, an iron (III)-ligand complex in contact with the working electrode, and an outlet for releasing the nitric oxide generated from the medium.

[0138] In some embodiments, the ligand forming the iron (III)-ligand complex is an iron complexing agent. The iron complexing agent may have one, two or more binding sites with the iron ion. In some embodiments, the iron complexing agent is an iron (III) complexing agent. In some embodiments, the iron complexing agent contains one or more nitrogen atoms. In some embodiments, the iron complexing agent contains one or more oxygen atoms. In some embodiments, the iron complexing agent contains both nitrogen atoms and oxygen atoms.

[0139] In some embodiments, the iron (III)-ligand complex of the present disclosure is soluble in a medium having a pH of 5 to 9. In some embodiments, the solubility in the medium is at least 0.1 mM. In some embodiments, the solubility in the medium is at least 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, or 50 mM. In some embodiments, when the pH is 5.5 to 9, or 5.5 to 8.5, 6.5 to 8.2 or 6.8 to 8.0, the solubility in the medium is at least 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM or 50 mM, but is not limited thereto.

[0140] In some embodiments, the iron (III)-ligand complexes of the present disclosure are stable at temperatures of 30° C. or more and / or below 95° C., 90° C., 85° C., or 80° C. In some embodiments, the iron (III)-ligand complexes of the present disclosure are stable at temperatures of 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., or 75° C. or more.

[0141] In some embodiments, the iron (III)-ligand complex is stable in a medium under certain conditions, meaning that the complex does not degrade and / or form a precipitate in the medium for at least 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 1 day, 2 days, 1 week, 2 weeks, 1 month, 2 months, or 6 months. In some embodiments, the conditions include pH levels and / or temperatures as described above. For example, the pH can be 5 to 9, 5.5 to 8.5, 6.5 to 8.2, or 6.8 to 8.0, but is not limited thereto. In some embodiments, the temperature is 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, or 75°C or higher.

[0142] Table A provides non-limiting examples of iron complexing agents (ligands) and corresponding iron-ligand complexes. In a specific embodiment, the iron complexing agent (ligand) is nitrilotriacetic acid (NTA) or a salt thereof. In another embodiment, the iron complexing agent (ligand) is 8-hydroxyquinoline-5-sulfonic acid (HQSA) or a salt thereof. In another embodiment, the iron complexing agent (ligand) is citric acid (CA) or a salt thereof. In another embodiment, the iron complexing agent (ligand) is N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) or a salt thereof.

[0143] In one embodiment, the iron(III)-ligand complex is In one embodiment, the iron(III)-ligand complex is In one embodiment, the iron(III)-ligand complex is In one embodiment, the iron(III)-ligand complex is

[0144] In some embodiments, the iron (III)-ligand complex is provided in a medium in which electrolysis is performed. In some embodiments, the iron (III)-ligand complex is present in the medium at a concentration of 0.1 to 50.0 mM. In some embodiments, the iron (III)-ligand complex is present in a concentration of 0.5 to 50.0 mM, or 1.0 to 50.0 mM, 2.0 to 40.0 mM, 5.0 to 50.0 mM, 5.0 to 40.0 mM, 5.0 to 35.0 mM, 5.0 to 30.0 mM, 10.0 to 50.0 mM, 10.0 to 40.0 mM, 10.0 to 30.0 mM, or 10.0 to 25.0 mM. In some embodiments, the iron (III)-ligand complex is present at a concentration of at least 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM or 40 mM. In some embodiments, the iron (III)-ligand complex is present at a concentration of no greater than 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM or 10 mM.

[0145] In some embodiments, the iron (III)-ligand complex is attached or otherwise immobilized to the working electrode while in contact with the medium. For example, immobilization may require that the iron (III)-ligand complex is covalently attached to the surface of the working electrode. In another example, the iron (III)-ligand complex can be physically adsorbed to the working electrode, or doped or covalently attached to a polymer, film, or hydrogel deposited on the surface of the working electrode.

[0146] The medium comprising nitrite ions is used as an electrolyte in the electrochemical process. In some embodiments, the medium comprises at least a suitable buffer (or buffering agent) to provide and / or maintain a desired pH.

[0147] In some embodiments, the buffer can be any commonly used buffer, as long as it does not interfere with the stability or solubility of the iron (III)-ligand complex. Many such useful buffers are known, and non-limiting examples include 4-hydroxyethylpiperazineethanesulfonic acid, 3-morpholinepropanesulfonic acid, tris, diethylbarbiturate, glycine-hydrochloric acid, phosphate-borax, phosphate, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, tris(hydroxymethyl)aminomethane, N-tris(hydroxymethyl)methylglycine, boric acid-borax, and combinations thereof.

[0148] The buffer may have a concentration suitable for providing a desired pH. In one embodiment, the concentration is 0.01 to 5.0 M. In one embodiment, the concentration is 0.02 to 2.0 M, 0.05 to 1.0 M, 0.1 to 0.9 M, 0.2 to 0.8 M, or 0.3 to 0.7 M, but is not limited thereto.

[0149] In some embodiments, the medium includes a nitrite source, which can be an inorganic nitrate or an organic nitrite. The inorganic nitrate can be NO2 - Examples include, but are not limited to, nitrites of Li, Na, K, Rb, Ca, Mg, Al, and Fe. In one embodiment, the nitrate is sodium nitrite (NaNO2). Examples of organic nitrites include R4N + NO2 - 、RNH3 + NO2 - , nicleosamide, nifedipine, flutamine, isobutyl nitrate, isopentyl nitrite, 1,3-propane nitrite, 1,5-pentane nitrite, 1,7-heptane nitrite and cyclohexylmethyl nitrite, but are not limited thereto.

[0150] The nitrite ions may be present in the medium at a concentration of at least 0.01 M. In some embodiments, the concentration is 0.01 to 20.0 M. In some embodiments, the concentration is 0.02 to 10.0 M. In some embodiments, the concentration is 0.05 to 7.0 M. In some embodiments, the concentration is 0.05 to 5.0 M. In some embodiments, the concentration is 0.1 to 4.0 M, 0.2 M to 2 M, 0.5 M to 1.0 M, 0.5 to 7.0 M, 0.5 to 6.0 M, 0.5 to 5.0 M, 0.5 to 4.0 M, 0.5 M to 3 M, 0.5 M to 2.0 M, or 0.5 M to 1.5 M, but are not limited thereto.

[0151] In some embodiments, an inlet for delivering a carrier gas to the medium is also included. The carrier gas may include any suitable gas, such as air, nitrogen, helium, argon and oxygen. In some embodiments, the carrier gas includes nitrogen. In some embodiments, the nitrogen concentration in the carrier gas is about or higher than 99.0% by volume. For example, the nitrogen concentration in the carrier gas may be about or higher than 99.10%, 99.20%, 99.30%, 99.40%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 99.95%, 99.98% or 99.99% by volume. In some embodiments, the carrier gas comprises oxygen. For example, the oxygen concentration in the carrier gas may be less than about 1%, 0.5% or 0.1%.

[0152] In some embodiments, the inlet is configured to sweep the carrier gas across the surface of the working electrode. Sweeping the surface of the electrode can increase the Faradaic efficiency and / or the reaction rate of the electrochemical reaction at and / or near the electrode surface and / or can increase the nitric oxide concentration of the product gas.

[0153] Example

[0154] The following examples are included to illustrate the specific embodiments of the present disclosure. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that can function well in the practice of the present disclosure, and therefore can be considered to constitute a specific mode of its practice. However, it will be appreciated by those skilled in the art based on the present disclosure that many changes can be made to the disclosed specific embodiments, and still obtain the same or similar results, without departing from the spirit and scope of the present disclosure.

[0155] Materials and methods

[0156] The electrolyte was prepared as follows:

[0157] Step 1. Prepare NaNO2 solution in HEPES buffer and adjust the solution to the desired pH using NaOH solution;

[0158] Step 2. Slowly dissolve the desired amount of ligand in the Fe2(SO4)3 solution and stir until completely dissolved;

[0159] Step 3. Add the Fe2(SO4)3 solution prepared in step 2 to the NaNO2 solution in step 1; and stir the resulting mixture to obtain an electrolyte.

[0160] Nitric oxide generation test was performed using a device made according to the description in PCT application publication number WO 2022127902A1. Concentration of nitric oxide at different currents.

[0161] Example 1. Trisodium nitrilotriacetate (NTA) as a ligand

[0162] This example tests the performance of the complex Fe-trisodium nitrilotriacetate (NTA). NTA has the following structure:

[0163]

[0164] The Fe-ligand complex has the following structure:

[0165]

[0166] The electrolyte used contained 5 mM Fe2(SO4)3, 20 mM NTA, 1.0 M NaNO2, 0.5 M Hepes, pH 7.01. The nitric oxide concentration was measured at a flow rate of 2 L / min at currents of 40 mA, 20 mA, and 10 mA, respectively.

[0167] The measured NO concentration is Figure 1A As shown. Figure 1B As shown, there is a good correlation between the current and NO concentration (R 2 =0.9953). The Faradaic efficiency (FE%) of the system is calculated as follows:

[0168]

[0169] Among them C NO is the concentration of nitric oxide (in ppm), Q is the flow rate (in L / min), n is the number of electrons transferred (here 1), F is the Faraday constant (96485C / mol), I is the operating current (in A), t is the time (in seconds), and Vm is the gas molar volume calculated based on temperature and pressure (pV=nRT). The calculated Faraday efficiency is shown in Table 1.

[0170] Table 1. Faradaic efficiency at different current levels

[0171] Current (mA) 40 20 10 FE% 34.40 42.59 51.11

[0172] Example 2. 8-Hydroxyquinoline-5-sulfonic acid (HQSA) as a ligand

[0173] This example tests the performance of the complex Fe-8-hydroxyquinoline-5-sulfonic acid (HQSA). HQSA has the following structure:

[0174]

[0175] The Fe-ligand complex has the following structure:

[0176]

[0177] The electrolyte used contained 5 mM Fe2(SO4)3, 30 mM HQSA, 1.0 M NaNO2, 0.5 M Hepes, pH 6.82. The nitric oxide concentration was measured at a current of 40 mA and 20 mA at a flow rate of 2 L / min.

[0178] The measured NO concentration is Figure 2 The calculated Faraday efficiency is shown in Table 2.

[0179] Table 2. Faradaic efficiency at different current levels

[0180] Current (mA) 40 20 FE% 48.16 71.42

[0181] Example 3. Citric acid (CA) as a ligand

[0182] This example tests the performance of the complex Fe-citric acid (CA). CA has the following structure:

[0183]

[0184] The Fe-ligand complex has the following structure:

[0185]

[0186] The electrolyte used contained 10 mM Fe2(SO4)3, 20 mM citric acid, 1.0 M NaNO2, 0.5 M Hepes, pH 7.4. The nitric oxide concentration was measured at a flow rate of 2 L / min at currents of 40 mA, 20 mA, and 10 mA, respectively.

[0187] The measured NO concentration is Figure 3A As shown. Figure 3B As shown, there is a good correlation between the current and NO concentration (R 2 =0.9924).

[0188] The calculated Faradaic efficiencies are shown in Table 3.

[0189] Table 3. Faradaic efficiency at different current levels

[0190] Current (mA) 40 20 10 FE% 77.32 89.77 89.11

[0191] Example 4. N-(2-Hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) as a ligand

[0192] This example tests the performance of the complex Fe-N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA). HEDTA has the following structure:

[0193]

[0194] The Fe-ligand complex has the following structure:

[0195]

[0196] The electrolyte used contained 5 mM Fe2(SO4)3, 10 mM HEDTA, 1.0 M NaNO2, 0.5 M Hepes, pH 7.3. The nitric oxide concentration was measured at a flow rate of 2 L / min at currents of 40 mA, 20 mA, and 30 mA, respectively.

[0197] The measured NO concentration is Figure 4 The calculated Faraday efficiency is shown in Table 4.

[0198] Table 4. Faradaic efficiency at different current levels

[0199] Current (mA) 40 30 20 FE% 31.12 29.70 38.66

[0200] Comparative Example 1

[0201] In this comparative example, porphyrin was used as a ligand. The Fe-ligand complex has the following structure:

[0202]

[0203] The electrolyte used contained 5 mM Fe-porphyrin, 1.0 M NaNO2, 0.5 M Hepes, pH 7.05. The nitric oxide concentration was measured at a current of 10 mA and 20 mA at a flow rate of 2 L / min.

[0204] The measured NO concentration is Figure 5 The actual performance is not only inferior to that tested in Examples 1-4, but also inferior to the copper (II) ligand complex.

[0205] Comparative Example 2

[0206] In this comparative example, N,N',N"-trimethyl-1,4,7-triazacyclononane (Me3TACN) was used as a ligand.

[0207]

[0208] Unfortunately, the preparation of the electrolyte (7 mM Fe-Me3TACN, 1.0 M NaNO2, 0.5 M Hepes, pH = 6.99) resulted in a large amount of orange precipitate. The experiment was not continued.

[0209] Comparative Example 3

[0210] In this comparative example, Cu-citric acid was used instead of Fe-ligand complex. The Cu-ligand complex has the following structure:

[0211]

[0212] The electrolyte used contained 10 mM Cu-CA, 1.0 M NaNO2, 0.5 M Hepes, pH 7.01. Nitric oxide concentration was measured at 40 mA with a flow rate of 2 L / min.

[0213] The measured NO concentration is Figure 6The Faradaic efficiency at 40 mA is 9.34%, which is much lower than its Fe-ligand counterpart.

[0214] Example 5. Effect of different concentrations of nitrite ions

[0215] This example tests the effect of nitrite ion concentration on NO generation efficiency.

[0216] The electrolytes tested contained 15 mM Fe2(SO4)3, 60 mM citric acid (CA), 0.5 M Hepes (pH 7.0), and NaNO2 at concentrations of 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, or 7.0 M. Nitric oxide concentrations were measured at a current of 5 mA at a flow rate of 0.24 L / min.

[0217] The results are as follows Figure 8 Surprisingly, the NO generation efficiency was highest at about 5.0 M NaNO2, as higher concentrations (e.g., 6.0 M or 7.0 M) did not further improve the efficiency.

[0218] This example also tests the operating qualities of these electrolytes. It was observed that higher nitrite ion concentrations lowered the melting temperature (or solidification temperature) of the electrolyte ( Fig. 9 ), thereby allowing such electrolytes to have a wider range of operating conditions. Surprisingly, when the higher nitrite ion concentration is >5M, the higher nitrite ion concentration (<5M) leads to a higher melting temperature (or solidification temperature) of the electrolyte.

[0219] In subsequent experiments, the operation of an electrolyte containing 5.0 M NaNO2 was tested at two temperatures (25°C and -12°C) (other components remained unchanged). Surprisingly, the electrolyte performed essentially the same at these two very different temperatures ( Fig.10 ).

[0220] Example 6. Effect of nitrite ion concentration using other ligands

[0221] In Example 5, the effect of nitrite ion concentration was tested in an electrolyte using citric acid (CA) as a ligand. In this example, CA was replaced with HEDTA.

[0222] The electrolyte used contained 5 mM Fe2(SO4)3, 10 mM N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), 0.5-7 M NaNO2, 0.5 M HEPES, pH 7.0. The nitric oxide concentration was measured at a current of 5 mA and a flow rate of 300 L / min. Results ( Fig.11) showed that, like CA, when HEDTA was used as a ligand, good NO generation efficiency was observed when the NaNO2 concentration was between 0.5 and 7 M.

[0223] ***

[0224] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0225] The invention illustratively described herein may be suitably implemented in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be interpreted broadly and not in a limiting sense. Furthermore, the terms and expressions used herein have been used as terms of description, not of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described, or portions thereof, but it is recognized that various modifications may be made within the scope of the claimed invention.

[0226] Therefore, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may make modifications, improvements and variations to the invention embodied therein disclosed herein, and such modifications, improvements and variations are considered to be within the scope of the present invention. The materials, methods and examples provided herein represent preferred embodiments, are exemplary, and are not intended to limit the scope of the present invention.

[0227] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether the removed material is specifically recited herein.

[0228] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0229] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if individually incorporated by reference. In the event of a conflict, the present specification, including definitions, will control.

[0230] It should be understood that although the present disclosure has been described in conjunction with the above embodiments, the foregoing description and examples are intended to illustrate rather than limit the scope of the present disclosure. Other aspects, advantages and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure belongs.

Claims

1. A device for generating nitric oxide, comprising: Working electrode and reference or counter electrode, a medium in contact with the working electrode and the reference or counter electrode, wherein the medium comprises nitrite ions, an iron(III)-ligand complex in contact with the working electrode, and An outlet for releasing nitric oxide generated from the medium.

2. The device of claim 1, wherein the ligand is an oxygen-containing or nitrogen-containing iron complexing agent.

3. The device of claim 1, wherein the iron (III)-ligand complex is stable at 40-95°C for at least 1 hour.

4. The device of claim 1, wherein the ligand is selected from Table A.

5. The device according to claim 4, wherein the ligand is selected from the group consisting of nitrilotriacetic acid (NTA) or a salt thereof, 8-hydroxyquinoline-5-sulfonic acid (HQSA) or a salt thereof, citric acid (CA) or a salt thereof, and N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) or a salt thereof.

6. The device according to claim 5, wherein the iron (III)-ligand complex is selected from the group consisting of:

7. The device according to any one of the preceding claims, wherein the iron (III)-ligand complex is present in a concentration of 1.0 to 70.0 mM.

8. The device according to any of the preceding claims, wherein the pH of the medium is from 5 to 9, or preferably from 5.5 to 8.5, from 6.5 to 8.2, or from 6.8 to 8.

0.

9. The device of claim 8, wherein the medium comprises a buffer.

10. The device according to claim 9, wherein the buffer comprises a reagent selected from the group consisting of: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof, 3-(N-morpholino)propanesulfonic acid (MOPS) or a salt thereof, tris(hydroxymethyl)aminomethane (Tris) or a salt thereof, barbiturate or a salt thereof, phosphoric acid or a salt thereof, boric acid or a salt thereof, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) or a salt thereof, N-tris(hydroxymethyl)methylglycine (Tricine) or a salt thereof, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TES) or a salt thereof. APS or a salt thereof, N,N-bis(2-hydroxyethyl)glycine (Bicine) or a salt thereof, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) or a salt thereof, dimethylarsonic acid or a salt thereof, citric acid or a salt thereof, 2-(N-morpholino)ethanesulfonic acid (MES) or a salt thereof, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) or a salt thereof, 2,2-bis(hydroxyethyl)-(iminotris)-(hydroxymethyl)-methane (Bis-Tris) or a salt thereof, glycine-hydrochloric acid, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl), and a combination thereof.

11. The device according to claim 9 or 10, wherein the concentration of the buffer is 0.05 to 1.5M.

12. The device according to any one of the preceding claims, wherein the nitrite ions are provided as inorganic or organic nitrite.

13. The device of claim 12, wherein the nitrite ions are provided as NaNO2.

14. The device according to claim 12 or 13, wherein the concentration of the nitrite ions is 0.05 to 7.0M.

15. Apparatus according to any preceding claim, further comprising an inlet for delivering a carrier gas into the medium.

16. A method for producing nitric oxide, comprising electrolyzing an iron (III)-ligand complex in a medium containing nitrite ions to produce nitric oxide, wherein: The pH of the medium is between 5 and 9, and The iron (III)-ligand complex is soluble and stable in the medium at a temperature of 40-95° C. for at least 1 hour.

17. The method of claim 16, wherein the iron (III)-ligand complex is soluble and stable in the medium when the pH of the medium is 5.5 to 8.5, 6.5 to 8.2 or 6.8 to 8.

0.

18. The method of claim 16 or 17, wherein the ligand is an oxygen-containing or nitrogen-containing iron complexing agent.

19. The method according to any one of claims 16 to 18, wherein the ligand is selected from Table A.

20. The method of claim 19, wherein the ligand is selected from the group consisting of nitrilotriacetic acid (NTA) or a salt thereof, 8-hydroxyquinoline-5-sulfonic acid (HQSA) or a salt thereof, citric acid (CA) or a salt thereof, and N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) or a salt thereof.

21. The method of claim 20, wherein the iron (III)-ligand complex is selected from the group consisting of:

22. The method according to any one of claims 16 to 21, wherein the iron (III)-ligand complex is present at a concentration of 1.0 to 50.0 mM.

23. The method according to any one of claims 16 to 22, wherein the medium comprises a buffer.

24. The method according to claim 23, wherein the buffer comprises a reagent selected from the group consisting of: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof, 3-(N-morpholino)propanesulfonic acid (MOPS) or a salt thereof, tris(hydroxymethyl)aminomethane (Tris) or a salt thereof, barbiturate or a salt thereof, phosphoric acid or a salt thereof, boric acid or a salt thereof, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) or a salt thereof, N-tris(hydroxymethyl)methylglycine (Tricine) or a salt thereof, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TES) or a salt thereof. APS or a salt thereof, N,N-bis(2-hydroxyethyl)glycine (Bicine) or a salt thereof, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) or a salt thereof, dimethylarsonic acid or a salt thereof, citric acid or a salt thereof, 2-(N-morpholino)ethanesulfonic acid (MES) or a salt thereof, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) or a salt thereof, 2,2-bis(hydroxyethyl)-(iminotris)-(hydroxymethyl)-methane (Bis-Tris) or a salt thereof, glycine-hydrochloric acid, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl), and a combination thereof.

25. The method of claim 23 or 24, wherein the concentration of the buffer is 0.05 to 1.0 M.

26. A method according to any one of claims 16 to 25, wherein the nitrite ions are provided as inorganic or organic nitrite.

27. The method of claim 26, wherein the nitrite ions are provided as NaNO2.

28. The method according to claim 26 or 27, wherein the concentration of the nitrite ions is 0.05 to 7.0M.

29. The method of any one of claims 16 to 28, further comprising delivering a carrier gas into the medium to carry nitric oxide generated from the medium out of the medium.

30. The method of any one of claims 16 to 29, further comprising regulating the production of nitric oxide by regulating the potential or current applied to the electrolysis.

31. The method of claim 29 or 30, further comprising regulating the generation of nitric oxide by adjusting the flow rate of the carrier gas.

32. The method of any one of claims 16 to 31, further comprising regulating the production of nitric oxide by regulating the contact area between the iron (III)-ligand complex and a working electrode used to apply electrolysis.

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