WO2011062864A2

WO2011062864A2 - Inhibitors of nox enzymes and methods of use thereof

Info

Publication number: WO2011062864A2
Application number: PCT/US2010/056692
Authority: WO
Inventors: John David Lambeth; Susan M. Smith; Tsukasa Kawahara; Jaeki Min; James Snyder; Aiming Sun; Thota Ganesh
Original assignee: Emory University
Priority date: 2009-11-17
Filing date: 2010-11-15
Publication date: 2011-05-26
Also published as: WO2011062864A3

Abstract

Inhibitors of Nox enzymes, such as Noxl, Nox2, and Nox3, and methods of making and using such compounds are describe herein.

Description

INHIBITORS OF NOX ENZYMES AND METHODS OF USE THEREOF

FIELD

The invention is in the field of small molecule inhibitors of NADPH-oxidase (Ni enzymes and methods of making and using thereof.

BACKGROUND

The NADPH-oxidase (Nox) enzymes represent a family of seven membrane enzymes, Noxl , Nox, 2, Nox3, Nox4, Nox 5, Duoxl , and Duox2, which catalyze NADPH-dependent generation of superoxide and secondary reactive oxygen species. While these enzymes have normal biological functions in signal transduction and host defense, they have also been implicated in the pathogenesis of a variety of diseases and disorders. Currently, no isoform- or class-selective Nox inhibitors have been approved. Potential inhibitors that have been investigated, such as diphenylene iodonium (DPI), apocynin, Nox2 B-loop peptide,

VAS2870, and pyrazolopyridines are ineffective and/or cause adverse side effects. There exists a need for inhibitors for Nox enzymes, such as Noxl , Nox2, and Nox3.

SUMMARY

Inhibitors of Nox enzymes, such as Noxl , Nox2, and Nox3, and methods of making and using such compounds are describe herein. In one embodiment, the compound has one the following formulas:

wherein, Emory Ref.: 10004 PCT

A is a monocyclic or bicyclic aromatic or non-aromatic ring having 5-12 atoms in the ring, wherein the ring optionally has one or more heteroatoms selected from C, O, S, N, and combinations thereof;

Ri is selected from the group consisting of hydrogen, hydroxyl, halogen, alkyl, alkoxy, aryl, alkylaryl, heteroaryl, alkylheteroaryl, heterocyclyl, and alkylheterocyclyl;

R₂ is selected from the group consisting of halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, ~CF₃, and --CN;

X is O, S, or N-R₃, wherein R₃ is OH, alkyl, or substituted or unsubstituted aryl; and

Y is C, O, N, N-R₄, S and Se;

Formula II

wherein,

Q is alky, hydroxy, ether, ester, carboxylic acid, N-R₄, S-R5, and Se, wherein R₄ is H, alkyl, or L-glutathione; Emory Ref.: 10004 PCT

Formula III

wherein,

X is C, O, N, and N-R₄, S, and Se; and

Y is O or N-R₃, wherein R₃ is OH, alkyl, or aryl; and

Z is CH, CH₂, or N; and

Formula IV

wherein,

Ri is selected from the group consisting of hydrogen, hydroxyl, halogen, alkyl, alkoxy, aryl, alkylaryl, heteroaryl, alkylheteroaryl, heterocyclyl, and alkylheterocyclyl; Emory Ref.: 10004 PCT

Y is C, O, N, N-R4, S or Se.

In certain embodiments, X is O.

In certain embodiments, Y is S, N, or Se or Y is S or N.

In certain embodiments, A is substituted or unsubstituted phenyl or the phenyl group is substituted with one or more halogen atoms or the phenyl group is substituted with one or more fluorine atoms.

In certain embodiments, Ri is aryl optionally substituted with one or more substituents or R¹ is substituted or unsubstituted phenyl, thiazolyl, or thiadiazolyl. In certain embodiments R¹ is aryl optionally substituted with one or more substituents.

In certain embodiments R¹ is aryl or heteroaryl, wherein the aryl or heteroaryl are optionally substituted with alkyl, alkoxy, halogen, cyano, alkylthio, hydroxyalkyl, alkanoyl, alkylcarbonate ester, alkyl carbamoyl amide, aryl, or heterocyclyl wherein these substituents are optionally substituted with alkyl, alkoxy, halogen, cyano, alkylthio, hydroxyalkyl, alkanoyl, alkylcarbonate ester, alkyl carbamoyl amide, aryl, or heterocyclyl.

In certain embodiments, Q is selected from the group consisting of alky, hydroxy, ether, ester, carboxylic acid, N-R⁴, S-R⁵, and Se, wherein R⁴ is H or alkyl and R⁵ is H, alkyl, or L-glutathione.

In certain embodiments, the invention relates to the use of a compound as described herein in the production of a medicament for the treatment of a NOX related disease.

Compounds disclosed here can be contained in pharmaceutical compositions and

administered alone or in combination with one or more additional active agents. The active agents can be administered simultaneously in the same dosage form or in separate dosage forms. Alternatively, the active agents can be administered sequentially in different dosage forms. Emory Ref.: 10004 PCT

The compound can be combined with one or more pharmaceutically acceptable excipients to form a pharmaceutical composition. The compositions can be formulated for enteral, parenteral, topical, transdermal, or pulmonary administration. The compounds can be formulated for immediate release, controlled release, and combinations thereof. Examples of controlled release formulations include delayed release, extended release, pulsatile release, and combinations thereof.

The compounds described herein can be used to treat a variety of Nox-related diseases including, but not limited to, hypertension, chronic obstructive pulmonary disease (COPD), Alzheimer's disease (AD), Parkinson's disease (PD), acute respiratory distress syndrome (ARDS), acute lung injury (ALI), amyotrophic lateral sclerosis (ALS), atherosclerosis, aging- related deafness, inflammatory diseases, such as arthritis; various cancers such as colon cancer, prostate cancer, fibrotic diseases, such as liver fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic systemic fibrosis, Crohn's disease, and

scleroderma/systemic sclerosisreper, reperfusion injury-related disorders, such as myocardial infarction; ischemic stroke, preservation of organs during transplantation,

ischemia/reperfusion injury (including stroke, myocardial infarction), diabetes, acute lung inflammation, cardiac hypertrophy, diabetic nephropathy, scar formation, skin aging and damage, and psoriasis.

In some embodiments, it is contemplated that compositions disclosed herein can be administered to subject before, during or after certain medical procedures, such as, organ transplants (heart, kidneys, liver, lungs, pancreas, intestine, and thymus) or other surgeries that reduce blood flow (cardiovascular surgery). The subject may be receiving or donating the organ.

In some embodiments, it is contemplated that composition disclosed herein can be used in biological (organ, tissue, or cell) storage mediums, typically aqueous solutions maintained at or below room temperatures, which may contain other ingredients such as, but not limited to, salts (sodium chloride, sodium lactate, calcium chloride, potassium chloride), amino acids, saccharides, polysaccharides (dextran, chondroitin, hydroxyethyl starch), vitamins (thiamine, ascorbic acid, calciferol, riboflavin, pyridoxine, tocopherol, cobalamins, Emory Ref.: 10004 PCT phylloquinone, pantothenic acid, biotin, niacin, folic acid) and/or adenosine triphosphate or precursors (adenosine, inosine, and adenine).

In certain embodiments, the invention relates to method of making compounds disclosed herein by mixing starting materials and reagents disclosed herein under conditions such that the compounds are formed.

DETAILED DESCRIPTION

I. Definitions

"Aryl", as used herein, refers aromatic, heterocyclic, fused aromatic, fused

heterocyclic, biaromatic, or bihetereo cyclic ring systems, optionally substituted with one or more substituents. Monocyclic aromatic ring systems generally contain 5, 6, or 7 ring atoms, while bicyclic and polycyclic systems generally contain 8-12 atoms. Broadly defined, "Ar", as used herein, includes single-ring or polycyclic, including bicyclic, aromatic groups that may include from zero to four heteroatoms, such as O, N, S, and combinations thereof.

Exemplary heteroaryl rings include, but are not limited to, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics". The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF₃, -CN, or the like. The term "Ar" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (i.e., "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples of

heterocyclic ring include, but are not limited to, benzimidazolyl, benzofuranyl,

benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,

benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H- 1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, Emory Ref.: 10004 PCT imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl,

octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-l,2,5-thiadiazinyl, 1,2,3- thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

"Alkyl", as used herein, refers to the radical of saturated or unsaturated aliphatic groups, including straight-chain alkyl, alkenyl, or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups, cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unless otherwise indicated, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

"Alkoxycarbonyl", as used herein, refers to a substituent having the following chemical formula:

wherein R is a linear, branched, or cyclic alkyl group, wherein j is from about 1 to about 12. Emory Ref.: 10004 PCT

"Alkoxycarbamido", as used herein, refers to a substituent having the following chemical formula:

wherein Rg is alkoxy and R 9 is hydrogen, alkoxy-alkyl, or alkanoyl, and j is from about 1 to about 12.

"Alkylaryl", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or hetero aromatic group).

"Heterocycle" or "heterocyclic", as used herein, refers to a cyclic radical attached via a ring carbon or nitrogen of a monocyclic or bicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ring atoms, consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (C\ - 4)alkyl, phenyl or benzyl, and optionally containing 1-3 double bonds and optionally substituted with one or more substituents. Examples of heterocyclic ring include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl,

benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l ,5,2-dithiazinyl,

dihydrofuro[2,3-¾]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl,

octahydroisoquinolinyl, oxadiazolyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, Emory Ref.: 10004 PCT quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-l ,2,5-thiadiazinyl, 1 ,2,3- thiadiazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,5-thiadiazolyl, 1 ,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

"Heteroaryl", as used herein, refers to a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and 1 , 2, 3, or 4 heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) where Y is absent or is H, O, (C \ -Cg) alkyl, phenyl or benzyl. Non-limiting examples of heteroaryl groups include furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide), quinolyl (or its N-oxide) and the like. The term "heteroaryl" can include radicals of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto. Examples of heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl (or its N-oxide), thientyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N- oxide), quinolyl (or its N-oxide), and the like.

"Halogen", as used herein, refers to fluorine, chlorine, bromine, or iodine.

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

The terms ortho, meta and para apply to 1 , 2-, 1 ,3- and 1 ,4-disubstituted benzenes, respectively. For example, the names 1 ,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

"Pharmaceutically acceptable salt", as used herein, refer to derivatives of the compounds defined by Formula I and II wherein the parent compound is modified by making acid or base salts thereof. Example of pharmaceutically acceptable salts include but are not limited to mineral or organic acid salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Such Emory Ref.: 10004 PCT conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acids; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, tolunesulfonic, naphthalenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic salts.

The pharmaceutically acceptable salts of the compounds can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704; and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," P. Heinrich Stahl and Camille G. Wermuth, Eds., Wiley-VCH, Weinheim, 2002.

As generally used herein "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications

commensurate with a reasonable benefit/risk ratio.

"Prodrug", as used herein, refers to a pharmacological substance (drug) which is administered in an inactive (or significantly less active) form. Once administered, the prodrug is metabolized in the body (in vivo) into the active compound. II. Compounds

A. Nox inhibitors

Inhibitors of Nox enzymes, such as Noxl, Nox2, and Nox3, are described herein. In one embodiment, the compound has one of the following formulas: Emory Ref.: 10004 PCT

Formula I wherein

X is O, S, or N-R₃, wherein R3 is OH, alkyl, or substituted or unsubstituted aryl; and

Y is C, O, N, N-R4, S and Se;

Formula II

Q is alky,hydroxy, ether, ester, carboxylic acid, N-R4, S-R5, and Se, wherein R4 is H, alkyl, or L-glutathione;

Formula III

X is C, O, N, and N-R₄, S, and Se; and

Y is O or N-R₃, wherein R₃ is OH, alkyl, or aryl; and

Z is CH, CH₂, or N;

Formula IV Emory Ref.: 10004 PCT

Y is C, O, N, N-R4, S and Se.

The compound can be administered as the free acid or free base or a pharmaceutically acceptable salt thereof. Alternatively, the compound can be administered as a prodrug or a pharmaceutically acceptable salt thereof.

The compounds described herein may have one or more chiral centers and thus exist as one or more stereoisomers. Such stereoisomers can exist as a single enantiomer, a mixture of diastereomers or a racemic mixture.

As used herein, the term "stereoisomers" refers to compounds made up of the same atoms having the same bond order but having different three-dimensional arrangements of atoms which are not interchangeable. The three-dimensional structures are called

configurations. As used herein, the term "enantiomers" refers to two stereoisomers which are non-superimposable mirror images of one another. As used herein, the term "optical isomer" is equivalent to the term "enantiomer". As used herein the term "diastereomer" refers to two stereoisomers which are not mirror images but also not superimposable. The terms

"racemate", "racemic mixture" or "racemic modification" refer to a mixture of equal parts of enantiomers. The term "chiral center" refers to a carbon atom to which four different groups are attached. Choice of the appropriate chiral column, eluent, and conditions necessary to effect separation of the pair of enantiomers is well known to one of ordinary skill in the art using standard techniques (see e.g. Jacques, J. et al, "Enantiomers, Racemates, and

Resolutions", John Wiley and Sons, Inc. 1981).

Exemplary Nox inhibitors are shown in the table below. Emory Ref.: 10004 PCT

Emory Ref.: 10004 PCT

Emory Ref.: 10004 PCT

Emory Ref.: 10004 PCT

In one embodiment, the compound is a compound of Formula I, wherein X is O, Y is S, N, or Se, A is a substituted or unsubstituted benzene ring, and Ri is a substituted or unsubstituted benzene ring, thiazole ring, or thiadiazole ring. In a particular embodiment, Y is O, X is S or N, preferably S, A is an unsubstituted benzene ring or is substituted with fluorine, and Ri is as defined above.

In another embodiment, the compound is a compound of Formula II, wherein X is O, A is a substituted or unsubstituted benzene ring, and Q is S or N. In a particular embodiment, the Q is a substituted thiol or amine.

III. Formulations

Depending upon the manner of introduction, the compounds described herein may be formulated in a variety of ways. Formulations containing one or more Nox inhibitors can be prepared in various pharmaceutical forms, such as granules, tablets, capsules, suppositories, powders, controlled release formulations, suspensions, emulsions, creams, gels, ointments, salves, lotions, or aerosols and the like. Preferably, these formulations are employed in solid dosage forms suitable for simple, and preferably oral, administration of precise dosages.

Solid dosage forms for oral administration include, but are not limited to, tablets, soft or hard gelatin or non-gelatin capsules, and caplets. However, liquid dosage forms, such as solutions, syrups, suspension, shakes, etc. can also be utilized. In another embodiment, the formulation is administered topically. Suitable topical formulations include, but are not limited to, lotions, ointments, creams, and gels. In a preferred embodiment, the topical formulation is a gel. In another embodiment, the formulation is administered intranasally.

A. Excipients, Carriers, and Additives.

Formulations containing one or more of the compounds described herein may be prepared using a pharmaceutically acceptable carrier composed of materials that are considered safe and effective and may be administered to an individual without causing Emory Ref.: 10004 PCT undesirable biological side effects or unwanted interactions. The carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. As generally used herein "carrier" includes, but is not limited to, diluents, binders, lubricants, disintegrators, fillers, pH modifying agents, preservatives, antioxidants, solubility enhancers, and coating compositions.

Carrier also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. Delayed release, extended release, and/or pulsatile release dosage formulations may be prepared as described in standard references such as "Pharmaceutical dosage form tablets", eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al, (Media, PA: Williams and Wilkins, 1995). These references provide information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients present in the drug-containing tablets, beads, granules or particles include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also referred to as

"fillers," are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium Emory Ref.: 10004 PCT chloride, dry starch, hydrolyzed starches, pregelatimzed starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.

Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatimzed starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatimzed starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross- linked PVP (Polyplasdone XL from GAF Chemical Corp).

Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.

Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium

dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, Emory Ref.: 10004 PCT propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenyl ether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl- .beta. -alanine, sodium N-lauryl-.beta.- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

If desired, the tablets, beads, granules, or particles may also contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, or preservatives.

The concentration of the Nox inhibitor(s) to carrier and/or other substances may vary from about 0.5 to about 100 wt.% (weight percent). For oral use, the pharmaceutical formulation will generally contain from about 5 to about 100% by weight of the active material. For other uses, the pharmaceutical formulation will generally have from about 0.5 to about 50 wt. % of the active material.

B. Modified release formulations

The compositions described herein can be formulation for modified or controlled release. Examples of controlled release dosage forms include extended release dosage forms, delayed release dosage forms, pulsatile release dosage forms, and combinations thereof.

Extended release dosage forms

The extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in "Remington - The science and practice of pharmacy" (20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000). A diffusion system typically consists of two types of devices, a reservoir and a matrix, and is well known and described in the art. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but are not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, cellulosic polymers such as methyl and ethyl cellulose, hydroxyalkylcelluloses Emory Ref.: 10004 PCT such as hydroxypropyl-cellulose, hydroxypropylmethylcellulose, sodium

carboxymethylcellulose, and Carbopol® 934, polyethylene oxides and mixtures thereof. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate and wax -type substances including hydrogenated castor oil or

hydrogenated vegetable oil, or mixtures thereof.

In certain preferred embodiments, the plastic material is a pharmaceutically acceptable acrylic polymer, including but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamine copolymer poly(methyl methacrylate), poly(methacrylic acid)(anhydride), polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), and glycidyl methacrylate copolymers.

In certain preferred embodiments, the acrylic polymer is comprised of one or more ammonio methacrylate copolymers. Ammonio methacrylate copolymers are well known in the art, and are described in NF XVII as fully polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.

In one preferred embodiment, the acrylic polymer is an acrylic resin lacquer such as that which is commercially available from Rohm Pharma under the tradename Eudragit®. In further preferred embodiments, the acrylic polymer comprises a mixture of two acrylic resin lacquers commercially available from Rohm Pharma under the tradenames Eudragit® RL30D and Eudragit ® RS30D, respectively. Eudragit® RL30D and Eudragit® RS30D are copolymers of acrylic and methacrylic esters with a low content of quaternary ammonium groups, the molar ratio of ammonium groups to the remaining neutral (meth)acrylic esters being 1 :20 in Eudragit® RL30D and 1 :40 in Eudragit® RS30D. The mean molecular weight is about 150,000. Edragit® S-100 and Eudragit® L-100 are also preferred. The code designations RL (high permeability) and RS (low permeability) refer to the permeability properties of these agents. Eudragit® RL/RS mixtures are insoluble in water and in digestive fluids. However, multiparticulate systems formed to include the same are swellable and permeable in aqueous solutions and digestive fluids.

The polymers described above such as Eudragit® RL/RS may be mixed together in any desired ratio in order to ultimately obtain a sustained-release formulation having a Emory Ref.: 10004 PCT desirable dissolution profile. Desirable sustained-release multiparticulate systems may be obtained, for instance, from 100% Eudragit® RL, 50% Eudragit® RL and 50% Eudragit® RS, and 10% Eudragit® RL and 90% Eudragit® RS. One skilled in the art will recognize that other acrylic polymers may also be used, such as, for example, Eudragit® L.

Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.

The devices with different drug release mechanisms described above can be combined in a final dosage form comprising single or multiple units. Examples of multiple units include, but are not limited to, multilayer tablets and capsules containing tablets, beads, or granules. An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using a coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.

Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation. Their formulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders.

Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils. Emory Ref.: 10004 PCT

Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In the congealing method, the drug is mixed with a wax material and either spray- congealed or congealed and screened and processed.

Delayed release dosage forms

Delayed release formulations are created by coating a solid dosage form with a polymer film, which is insoluble in the acidic environment of the stomach, and soluble in the neutral environment of the small intestine.

The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and/or enzymatically degradable polymers, and may be conventional "enteric" polymers. Enteric polymers, as will be appreciated by those skilled in the art, become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the

gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename Eudragit® (Rohm Pharma;

Westerstadt, Germany), including Eudragit® L30D-55 and L100-55 (soluble at pH 5.5 and above), Eudragit® L-100 (soluble at pH 6.0 and above), Eudragit® S (soluble at pH 7.0 and above, as a result of a higher degree of esterification), and Eudragits® NE, RL and RS (water- insoluble polymers having different degrees of permeability and expandability); vinyl Emory Ref.: 10004 PCT polymers and copolymers such as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene -vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multi-layer coatings using different polymers may also be applied.

The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.

The coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc. A plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt. % to 50 wt. % relative to the dry weight of the polymer. Examples of typical plasticizers include

polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides. A stabilizing agent is preferably used to stabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. % to 100 wt. % of the polymer weight in the coating solution. One effective glidant is talc. Other glidants such as magnesium stearate and glycerol monostearates may also be used. Pigments such as titanium dioxide may also be used. Small quantities of an anti-foaming agent, such as a silicone (e.g., simethicone), may also be added to the coating composition.

Pulsatile Release

The formulation can provide pulsatile delivery of the one or more Nox inhibitors. By "pulsatile" is meant that a plurality of drug doses are released at spaced apart intervals of time. Generally, upon ingestion of the dosage form, release of the initial dose is substantially immediate, i.e., the first drug release "pulse" occurs within about one hour of ingestion. This initial pulse is followed by a first time interval (lag time) during which very little or no drug is released from the dosage form, after which a second dose is then released. Similarly, a Emory Ref.: 10004 PCT second nearly drug release-free interval between the second and third drug release pulses may be designed. The duration of the nearly drug release-free time interval will vary depending upon the dosage form design e.g., a twice daily dosing profile, a three times daily dosing profile, etc. For dosage forms providing a twice daily dosage profile, the nearly drug release- free interval has a duration of approximately 3 hours to 14 hours between the first and second dose. For dosage forms providing a three times daily profile, the nearly drug release-free interval has a duration of approximately 2 hours to 8 hours between each of the three doses.

In one embodiment, the pulsatile release profile is achieved with dosage forms that are closed and preferably sealed capsules housing at least two drug-containing "dosage units" wherein each dosage unit within the capsule provides a different drug release profile. Control of the delayed release dosage unit(s) is accomplished by a controlled release polymer coating on the dosage unit, or by incorporation of the active agent in a controlled release polymer matrix. Each dosage unit may comprise a compressed or molded tablet, wherein each tablet within the capsule provides a different drug release profile. For dosage forms mimicking a twice a day dosing profile, a first tablet releases drug substantially immediately following ingestion of the dosage form, while a second tablet releases drug approximately 3 hours to less than 14 hours following ingestion of the dosage form. For dosage forms mimicking a three times daily dosing profile, a first tablet releases drug substantially immediately following ingestion of the dosage form, a second tablet releases drug approximately 3 hours to less than 10 hours following ingestion of the dosage form, and the third tablet releases drug at least 5 hours to approximately 18 hours following ingestion of the dosage form. It is possible that the dosage form includes more than three tablets. While the dosage form will not generally include more than a third tablet, dosage forms housing more than three tablets can be utilized.

Alternatively, each dosage unit in the capsule may comprise a plurality of drug- containing beads, granules or particles. As is known in the art, drug-containing "beads" refer to beads made with drug and one or more excipients or polymers. Drug-containing beads can be produced by applying drug to an inert support, e.g., inert sugar beads coated with drug or by creating a "core" comprising both drug and one or more excipients. As is also known, drug-containing "granules" and "particles" comprise drug particles that may or may not include one or more additional excipients or polymers. In contrast to drug-containing beads, Emory Ref.: 10004 PCT granules and particles do not contain an inert support. Granules generally comprise drug particles and require further processing. Generally, particles are smaller than granules, and are not further processed. Although beads, granules and particles may be formulated to provide immediate release, beads and granules are generally employed to provide delayed release.

C. Topical Formulations

In one embodiment, the compound is formulated for topical administration. Suitable topical dosage forms include lotions, creams, ointments, and gels. A "gel" is a semisolid system containing a dispersion of the active agent, i.e., Nox inhibitor, in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid may include a lipophilic component, an aqueous component or both. Some emulsions may be gels or otherwise include a gel component. Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components. Methods for preparing lotions, creams, ointments, and gels are well known in the art.

D. Other Active Agents

The Nox inhibitors described herein can be administered adjunctively with other active compounds. These compounds include but are not limited to analgesics, antiinflammatory drugs, antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine drugs, antimuscarinics, anxioltyics, sedatives, hypnotics, antipsychotics, bronchodilators, anti- asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastrointestinal drugs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, stimulants, anorectics and anti -narcoleptics. "Adjunctive administration", as used herein, means the Nox inhibitors can be administered in the same dosage form or in separate dosage forms with one or more other active agents.

Specific examples of compounds that can be adjunctively administered with the Nox inhibitors include, but are not limited to, aceclofenac, acetaminophen, adomexetine, almotriptan, alprazolam, amantadine, amcinonide, aminocyclopropane, amitriptyline, amolodipine, amoxapine, amphetamine, aripiprazole, aspirin, atomoxetine, azasetron, azatadine, beclomethasone, benactyzine, benoxaprofen, bermoprofen, betamethasone, bicifadine, bromocriptine, budesonide, buprenorphine, bupropion, buspirone, butorphanol, Emory Ref.: 10004 PCT butriptyline, caffeine, carbamazepine, carbidopa, carisoprodol, celecoxib, chlordiazepoxide, chlorpromazine, choline salicylate, citalopram, clomipramine, clonazepam, clonidine, clonitazene, clorazepate, clotiazepam, cloxazolam, clozapine, codeine, corticosterone, cortisone, cyclobenzaprine, cyproheptadine, demexiptiline, desipramine, desomorphine, dexamethasone, dexanabinol, dextroamphetamine sulfate, dextromoramide,

dextropropoxyphene, dezocine, diazepam, dibenzepin, diclofenac sodium, diflunisal, dihydrocodeine, dihydroergotamine, dihydromorphine, dimetacrine, divalproxex, dizatriptan, dolasetron, donepezil, dothiepin, doxepin, duloxetine, ergotamine, escitalopram, estazolam, ethosuximide, etodolac, femoxetine, fenamates, fenoprofen, fentanyl, fludiazepam, fluoxetine, fiuphenazine, flurazepam, flurbiprofen, fiutazolam, fiuvoxamine, frovatriptan, gabapentin, galantamine, gepirone, ginko bilboa, granisetron, haloperidol, huperzine A, hydrocodone, hydrocortisone, hydromorphone, hydroxyzine, ibuprofen, imipramine, indiplon,

indomethacin, indoprofen, iprindole, ipsapirone, ketaserin, ketoprofen, ketorolac, lesopitron, levodopa, lipase, lofepramine, lorazepam, loxapine, maprotiline, mazindol, mefenamic acid, melatonin, melitracen, memantine, meperidine, meprobamate, mesalamine, metapramine, metaxalone, methadone, methadone, methamphetamine, methocarbamol, methyldopa, methylphenidate, methylsalicylate, methysergid(e), metoclopramide, mianserin, mifepristone, milnacipran, minaprine, mirtazapine, moclobemide, modafinil (an anti-narcoleptic), molindone, morphine, morphine hydrochloride, nabumetone, nadolol, naproxen, naratriptan, nefazodone, neurontin, nomifensine, nortriptyline, olanzapine, olsalazine, ondansetron, opipramol, orphenadrine, oxaflozane, oxaprazin, oxazepam, oxitriptan, oxycodone, oxymorphone, pancrelipase, parecoxib, paroxetine, pemoline, pentazocine, pepsin, perphenazine, phenacetin, phendimetrazine, phenmetrazine, phenylbutazone, phenytoin, phosphatidylserine, pimozide, pirlindole, piroxicam, pizotifen, pizotyline, pramipexole, prednisolone, prednisone, pregabalin, propanolol, propizepine, propoxyphene, protriptyline, quazepam, quinupramine, reboxitine, reserpine, risperidone, ritanserin, rivastigmine, rizatriptan, rofecoxib, ropinirole, rotigotine, salsalate, sertraline, sibutramine, sildenafil, sulfasalazine, sulindac, sumatriptan, tacrine, temazepam, tetrabenozine, thiazides,

thioridazine, thiothixene, tiapride, tiasipirone, tizanidine, tofenacin, tolmetin, toloxatone, topiramate, tramadol, trazodone, triazolam, trifluoperazine, trimethobenzamide, trimipramine, Emory Ref.: 10004 PCT tropisetron, valdecoxib, valproic acid, venlafaxine, viloxazine, vitamin E, zimeldine, ziprasidone, zolmitriptan, Zolpidem, zopiclone and isomers, salts, and combinations thereof.

The additional active agent(s) can be formulated for immediate release, controlled release, or combinations thereof.

IV. Methods of Making

The Nox inhibitors can be prepared using methods known in the art. Exemplary synthetic methodologies are shown in Schemes A-C.

Scheme A

Emory Ref.: 10004 PCT

EDCI

Scheme C

The commercially available methyl thiosalicylate 2 can be reacted with various anilines using AlMe₃ -promoted amino lysis protocol to obtain substituted 2-mercaptoamides 3. 2-mercaptoamide 3 can be cyclized using hypervalent iodine reagent phenyliodine(III)bis- (trifluoroacetate) (PIFA) in CH₂CI₂ at 0°C in the presence of trifluoroacetic acid (TFA) to form the benzisothiazolone 4 (7, Scheme A). To modify the B ring of the benzisothiazolones, three different synthetic routes can be used as shown in Scheme B. The indazolones 10, which are the nitrogen analogs of the benzisothiazolones, can be prepared through a PIFA- mediated oxidative cyclization (6) shown in Scheme B. The selenium derivatives 16 were prepared from benzanilide using organolithiation as shown in Scheme B. The open form of the cyclic sulfur compounds were prepared by reacting Grignard reagents with

benzisothiazolones 4 to form the substituted thio amide 17 (1) shown in Scheme C.

V. Methods of Use

The compounds described herein can be used to treat a variety of Nox-related diseases including, but not limited to, hypertension, chronic obstructive pulmonary disease (COPD), Emory Ref.: 10004 PCT

Alzheimer's disease (AD), Parkinson's disease (PD), acute respiratory distress syndrome (ARDS), acute lung injury (ALI), amyotrophic lateral sclerosis (ALS), atherosclerosis, aging- related deafness, inflammatory diseases, such as arthritis; colon cancer, prostate cancer, fibrotic diseases, such as liver fibrosis, reperfusion injury-related disorders, such as myocardial infarction; ischemic stroke, and preservation of organs during transplantation.

As discussed above, Nox enzymes have been implicated in the pathogenesis of a variety of diseases and disorders. Nox enzymes exhibit several class-specific modes of regulation. Noxl, Nox2, and Nox3 represent a single regulatory class that is activated by regulatory subunits. Nox4 is constitutively active. Nox5, Duoxl, and Duox2 are activated by calcium. The compounds described herein have shown to inhibit the interaction of

Noxl/Nox2/Nox3 enzymes with their key regulatory subunits p47phox (mainly for Nox2) and NOXOl (for Noxl and Nox3). Specifically, p47phox and NOXOl are homologous proteins that contain a bis-SH3 type domain (SH3 domains are involved in protein-protein interactions and a bis-SH3 domain is a tandem repeat of two SH3 domains that function together to form a single binding site). The bis-SH3 domain binds to a pro line-rich domain (PRD) in the C- terminus of p22phox, a membrane protein that physically associates with Noxl, Nox2, and Nox3.

The amount of compound to be administered can be readily determined by the attending or prescribing physician. Dosages are dependent on a variety of factors including, but not limited to, disease or disorder to be treated and age and weight of the patient.

Exemplary dosages include, but are not limited to 0.001 mg/kg day to 100 mg/kg day.

Exemplary daily dosages include, but are not limited to 0.01 mg to 5000 mg per day, preferably 0.01 mg to 2000 mg per day, more preferably from 0.01 mg to 1000 mg per day, most preferably from 0.01 mg to 500 mg per day.

The compounds described herein can be administered once a day or multiple times a day, such as twice a day, three times a day, or four or more times a day. For controlled release compositions, the formulation may be administered once a day, every other day, once a week, once every two weeks, or once a month. Emory Ref.: 10004 PCT

Examples

Synthesis of Isothiazolone Analogs

A solution of methyl-4-aminobenzoate (E) (44 mmol, 2 eq) in dichlormethane (100 ml) was added drop wise a solution of trimethylaluminum (2M in tolune, (44 mmol, 2 eq) at 0 °C, then the reaction was brought to room temperature and stirred for 45 min. Methyl 2- mercaptobenzoate (22 mmol, 1 eq) in dichloromethane (10 ml) was introduced to the above reaction and then reaction mixture was brought to reflux overnight. Cooled the reaction and carefully quenched with 5% HC1 (50 mL). The white precipitate was filtered off through a celite plug. The filtrate was extracted with dichloromethane (2 x 20 ml). Combined organics were washed with saturated NaHC03, water, brine, and then dried with sodium sulfate and concentrated. The crude mass was on silica gel chromatography eluting with 10-50% ethyl acetate in hexane gave intermediate (40% yield). To a solution of the intermediate (2.8 mmol, 1 eq) in dichloromethane (20 ml) was added TFA (0.44 ml, 2 eq) at 0 °C. Then a solution of PIFA (2.8 mmol, 1 eq) in dichloromethane (30 ml) was introduced to the above reaction over 45 min. At the conclusion of the reaction by TLC, reaction mixture was concentrated to dryness. The crude mass was subjected to chromatography over silica gel column eluting with up to 0.5% methanol in dichloromethane to provide example 17 as a solid (540 mg, 66% yield).

Similarly other compounds listed in the disclosure including preferred examples 16, 18, 19, 23, 52, 53, and 54 are synthesized from the above synthetic methods. The compounds are characterized by NMR and MS data. Data for selected compounds is provided below.

Example 16: 1H NMR (400 MHz, CDC13) δ 8.10 (dd, J = 6.8, 2 Hz, 2H), 8.0 (d, J = 7.6 Hz, 1H), 7.86 (dd, J = 7, 1.6 Hz, 2H), 7.65 (t, J = 7.8 Hz, 1H), 7.56 (d, J = 8 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H). 3. 90 (s, 3H). LCMS. Cacld. for C15H12N03S (M+H) 286; found 286. Emory Ref.: 10004 PCT

Example 17: IH NMR (400 MHz, CDC13) δ 7.75 (dd, J = 8, 2.4 Hz, IH), 7.53 (d, J = 4 Hz, IH), 7.50 (d, J = 4 Hz, IH), 7.40 (t x d, J = 8.4, 2.4 Hz, IH), 7.15 (d, J = 2.4 Hz, IH), 7.01 (dd, J = 8.4, 2 Hz, IH), 6.85 (d, J = 8.4 Hz, IH) 6.0 (s, 2H). Anal. Calcd for C14H8FN03S: C, 58.13; H, 2.79; N, 4.84; S, 11.08; Found: C, 57.86; H, 2.72; N, 4.84, S. 11.28. LCMS. Cacld. for C14H9FN03S (M+H) 290; found 290.

Example 52: IH NMR (400 MHz, CDC13) δ 8.08 (m, 3H), 7.77 (t, J = 2 Hz, IH), 7.76 (t, J = 2 Hz, 2H), 7.65 (d, J = 3.6 Hz, 2H), 7.46 (m, IH), 3. 91 (s, 3H). LCMS. Cacld. for

C15Hl lN03Se (M+H) 333; found 333.

Example 53 : IH NMR (400 MHz, CDC13) δ 8.81 (dd, J=2.0Hz, 4.8Hz, IH), 8.34

(dd,J=1.6Hz, 8.0Hz, IH), 8.13 (d, J = 8.8Hz, 2H), 7.85 (d, J=8.8Hz, 2H), 7.41 (dd,J=4.8Hz, 8.0Hz, IH), 3.95 (s, 3H). LCMS. Cacld. for C14H10N2O3S (M+H) 287.0; found 287.0.

Example 54: IH NMR (400 MHz, CDC13) δ 8.19 (d, J=6.4Hz, IH), 8.12 (d,J=8.4Hz, 2H), 7.76 (d, J = 8.4Hz, 2H), 6.35 (d, J=6.4Hz, IH), 3.93 (s, 3H). LCMS. Cacld. for

CI 1H9N03S (M+H) 236.0; found 236.0. Synthesis of Selenanzolone Analogs 0₂CI

3-Fluorobenzoyl chloride (A) (3.8 mmol, 1.05 eq) was added drop wise to a solution of amine B (3.6 mmol, 1 eq), catalytic DMAP (20 mg) in dichloromethane (10 mL) at room temperature and then stirred for 3 hrs. The reaction mixture was concentrated and portioned between ethyl acetate and saturated NaHC03 solution (30 ml). Aqueous was extracted with more ethyl acetate. Combined organics were washed with water, brine, and then dried with sodium sulfate and concentrated. The crude mass was purified by silica gel chromatography Emory Ref.: 10004 PCT eluting with 10-15% ethyl acetate in hexane to provide intermediate C (90% yield). To a solution of C (1.2 mmol, 1 eq), in THF (5 ml) was added n-BuLi (2.71 mmol, 2.2 eq) drop wise at 0 oC and then stirred the reaction for 40 min at the same temperature. Se-powder (100 mg, 1 eq) was added and stirred the reaction mixture at 0 °C for 45 min. Then the reaction was cooled to -78 °C and was added CuBr2 (2.4 mmol, 2 eq) and then reaction was brought to room temperature overnight. 1% AcOH (5 mL) was added to quench the reaction followed by dichloromethane. Solution was filtered through celite pad, filtrate was concentrated to provide black gummy residue. This was subjected to chromatography over silica gel column eluting with 0.8% methanol in dichloromethane to provide example 48 as a yellow solid (150 mg, 36% yield).

Examples 47, 49, 50, 51 were similarly synthesized by the above synthetic methods.

Characterization data is provided below. Example 48: 1H NMR (400 MHz, CDC13) δ 7.9 (d, J = 8 Hz, 1H), 7.45 (m, 1H), 7.34 (t, J = 8 Hz, 1H), 7.1 (d, J = 2.4 Hz, 1H), 6.94 (dd, J = 8.2, 2 Hz, 1H), 6.83 (d, J = 8.4 Hz, 2H), 6.0 (s, 2H). Anal. Calcd for C14H8FN03Se: C, 50.02; H, 2.40; N, 4.17; Found: C, 50.13; H, 2.25; N, 4.16. LCMS. Cacld. for C14H9FN03Se (M+H) 338; found 338. Example 47: 1H NMR (400 MHz, CDC13) δ 7.85 (m, 1H), 7.30 (m, 1H), 7.06 (d, J = 2 Hz, 1H), 6.90 (dd, J = 8.4, 2.4 Hz, 1H), 6.82 (d, J = 8 Hz, 1H), 6.0 (s, 2H). LCMS. Cacld. for C14H8F2N03Se (M+H) 355; found 355.

Example 49: 1H NMR (400 MHz, CDC13) δ 7.89 (d, J = 7.6 Hz, 1H), 7.45 (m, 1H), 7.32 (t, J = 8 Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.6, 2.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 4.26 (s, 4H). LCMS. Cacld. for C15H1 lFN03Se (M+H) 352; found 352.

Example 50: 1H NMR (400 MHz, CDC13) δ 7.85 (d, J = 8.4 Hz, 1H), 7.62 (m, 3H), 7.41 (m, 2H), 7.20 (m, 2H), 4.06 (m, 2H), 3.97 (m, 2H). LCMS. Cacld. for C16H14N03Se (M+H) 348; found 348. Emory Ref.: 10004 PCT

Example 51 : 1H NMR (400 MHz, CDC13) δ 7.77 (d, J = 8.4 Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.90 (dd, J = 8.4, 2 Hz, 1H), 6.79 (d, J = 8 Hz, 1H), 5.97 (s, 2H), 3.96 (s, 6H). LCMS. Cacld. for C16H14N05Se (M+H) 380; found 380. Screening Assays

The compounds described herein were evaluated for cytotoxicity and ability to inhibit Noxl, Nox2, and Nox4.

Inhibition of Nox2 activity was determined using a cell-free assay in which isolated plasma membranes from human neutrophils were used as a source for Nox2-p22/?/zox, supplemented with recombinant p47phox, p67phox, and Rac 1 Q61 L (activated Rac), expressed in and isolated from E. coli, using L012 or luminol as a readout to monitor enzymatic activity.

Cell -based activity assays for Noxl, Nox2, and Nox4 were performed with transiently transfected cells expressing individual Nox/Duox isoenzymes (Noxl, Nox2, Nox3, or Nox4), along with their respective regulatory subunits (NOXOl, NOXAl, p47phox, and p67phox). L012 and luminol were used to monitor superoxide and hydrogen peroxide, respectively. Transfection and assay protocols are known in the literature.

The IC50 values for compounds disclosed herein for the FP binding assay and cell-free Nox2 assay are summarized in the Table below.

Noxl Cell-based Nox4 Cell-based

FP Assay Nox2 Cell free assay (inhibition assay (inhibition at

Examples

IC₅₀ (μΜ) IC50 (μΜ) at 10 μΜ) 10 μΜ)

ThrlOl 4 3 inhibits no inhibition

Thrl07 0.4 0.4 inhibits partial inhibition

1 2-3

2 5-6

3* 4-5

4* 0.9 1

5* inactive

6 inactive Emory Ref.: 10004 PCT * inactive

* inactive

9

0 1.7 3

* inactive

* 5-6

* 1.5 0.4

* 4

* 2.5

* 0.9 0.4 inhibits

7 0.6 0.3 inhibits

* 0.5 0.7 inhibits

* 0.3 0.6 inhibits

* 1.1

* 1 1.3

2 0.6 1 no inhibition

* 0.7 1 no inhibition

* 10

5 1 1

* 1 2

* 0.9

* inactive

* 1

* 2

1 3

2 inactive

3 4

4 0.6

5 4

* 22

7 inactive Emory Ref.: 10004 PCT

38 22

39 51

40* 19

41 inactive 1.3

42* inactive 0.7

43* inactive

44

45* inactive 4

46 inactive

47* 1.0 0.5

48* 0.2 0.5

49* 1.0 0.5

50* 0.3 0.9

51 * 1.0 3.0

52* 1.8 0.18

53* 1.0 1.2

Ebseien (Thr 07) ThrlOl

As shown above, several of the compounds in the table exhibited improved inhibition activity of the binding between p22phox PRD and bis-SH3 domain of p47phox as well as improved inhibition of Nox2 in a cell-free assay compared to ThrlOl . Several of the compounds in the table exhibit nanomolar potency for both the inhibition of the binding between p22phox PRD and bis-SH3 domain of p47phox and the inhibition of ROS generation in the Nox2 cell-free assay. In addition, the several of the compounds showed inhibition of Noxl .

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention Emory Ref.: 10004 PCT belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

Emory Ref.: 10004 PCT

A composition comprising a compound of formula I,

Formula I or a salt thereof wherein

A is a monocyclic or bicyclic aromatic or non-aromatic ring having 5-12 atoms in the ring or a double bond, wherein the ring optionally has one or more heteroatoms selected from O, S, or N;

Ri is aryl optionally substituted with one or more substituents;

R₂ is selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, ~CF₃, and --CN;

X is O, S, or N-R₃, wherein R₃ is OH, alkyl, or substituted or unsubstituted aryl;

Y is N-R₄, S or Se; and

R₄ is H or alkyl.

2. The composition of Claim 1, wherein R¹ is a substituted or unsubstituted phenyl, thiazolyl, or thiadiazolyl.

3. The composition of Claim 1, wherein X is O.

4. The composition of Claim 1, wherein A is an optionally substituted phenyl. Emory Ref.: 10004 PCT

2

5. The composition of Claim 1, wherein A is a phenyl group and R is

halogen atoms.

6. A compound of Claim 1 selected from:

2-phenylbenzo[d]isothiazol-3(2H)-one,

2-(4-methoxyphenyl)benzo[d]isothiazol-3(2H)-one,

2-(benzo[d][l,3]dioxol-5-yl)benzo[d]isothiazol-3(2H)-one,

2-(2,4-dimethylphenyl)benzo[d]isothiazol-3(2H)-one,

2-(4-fluorophenyl)benzo[d]isothiazol-3(2H)-one,

2-(2,4-dimethylphenyl)-5-fluorobenzo[d]isothiazol-3(2H)-one,

5-fluoro-2-(4-fluorophenyl)benzo[d]isothiazol-3(2H)-one,

2-(2-chloro-6-methylphenyl)-5-fluorobenzo[d]isothiazol-3(2H)-one,

5- fluoro-2-phenylbenzo[d]isothiazol-3(2H)-one,

2-(benzo[d][l,3]dioxol-5-yl)-5-fluorobenzo[d]isothiazol-3(2H)-one,

methyl 4-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzoate,

methyl 4-(5-fluoro-3-oxobenzo[d]isothiazol-2(3H)-yl)benzoate,

ethyl 4-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzoate,

tert-butyl 4-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzoate,

methyl 2-methoxy-4-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzoate,

methyl 3-chloro-4-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzoate,

4-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzonitrile,

methyl 2-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzoate,

2-(4-acetylphenyl)benzo[d]isothiazol-3(2H)-one,

2-(4-nitrophenyl)benzo[d]isothiazol-3(2H)-one,

2-(4-hydroxyphenyl)benzo[d]isothiazol-3(2H)-one,

methyl 6-(3-oxobenzo[d]isothiazol-2(3H)-yl)nicotinate,

6- (3-oxobenzo[d]isothiazol-2(3H)-yl)nicotinonitrile,

2-(4-(hydroxymethyl)phenyl)benzo[d]isothiazol-3(2H)-one,

2-benzylbenzo[d]isothiazol-3(2H)-one,

N-methyl-4-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzamide,

2-(4-hydroxyphenyl)benzo[d]isothiazol-3(2H)-one, Emory Ref.: 10004 PCT

2-(2,4-dimethylphenyl)-l-methyl-lH-indazol-3(2H)-one,

2-(4-fluorophenyl)- 1 -methyl- 1 H-indazol-3 (2H)-one,

2-(2,4-dimethylphenyl)-lH-indazol-3(2H)-one,

1 -methyl-2-phenyl- 1 H-indazol-3 (2H)-one,

2-(l,3,4-thiadiazol-2-yl)benzo[d]isothiazol-3(2H)-one,

2-(5-phenyl-l,3,4-thiadiazol-2-yl)benzo[d]isothiazol-3(2H)-one,

2-(5-(ethylthio)-l,3,4-thiadiazol-2-yl)benzo[d]isothiazol-3(2H)-one,

2-(5-(methylthio)-l,3,4-thiadiazol-2-yl)benzo[d]isothiazol-3(2H)-one,

5-fluoro-2-(l,3,4-thiadiazol-2-yl)benzo[d]isothiazol-3(2H)-one,

2-(5-(tert-butyl)-l,3,4-thiadiazol-2-yl)benzo[d]isothiazol-3(2H)-one,

2-(5-(4-bromophenyl)-l,3,4-thiadiazol-2-yl)benzo[d]isothiazol-3(2H)-one

2-(4-methylthiazol-2-yl)benzo[d]isothiazol-3(2H)-one,

2-(4,5-dimethylthiazol-2-yl)benzo[d]isothiazol-3(2H)-one,

2-(benzo[d][l,3]dioxol-5-yl)-4,5-difluorobenzo[d][l,2]selenazol-3(2H)-one,

2-(benzo[d][l,3]dioxol-5-yl)-5-fluorobenzo[d][l,2]selenazol-3(2H)-one,

2-(2,3-dihydrobenzo[b][l,4]dioxin-6-yl)-5-fluorobenzo[d][l,2]selenazol-3(2H)-

2-(4-(l,3-dioxolan-2-yl)phenyl)benzo[d][l,2]selenazol-3(2H)-one,

2-(benzo[d][l,3]dioxol-5-yl)-6,

7-dimethoxybenzo[d][l,2]selenazol-3(2H)-one, methyl 4-(3-oxobenzo[d][l,2]selenazol-2(3H)-yl)benzoate,

methyl 4-(3-oxoisothiazolo[5,4-b]pyridin-2(3H)-yl)benzoate, or

methyl 4-(3-oxoisothiazol-2(3H)-yl)benzoate.

A composition comprising a compound of formula II,

Formula II Emory Ref.: 10004 PCT

or a salt thereof wherein,

A is a monocyclic or bicyclic aromatic or non-aromatic ring having 5-12 atoms in the ring, wherein the ring optionally has one or more heteroatoms selected from O, S, N;

Ri is aryl optionally substituted with one or more substituents;

R₂ is selected from the group consisting of hydrogen, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties,— CF₃, and --CN;

Q is alky, hydroxy, ether, ester, carboxylic acid, N-R4, S-R₅, and Se, wherein R₄ is H, alkyl, and R⁵ is H, alkyl, or glutathione.

8. The composition of Claim 7, wherein R¹ is a substituted or unsubstituted phenyl, thiazolyl, or thiadiazolyl.

9. A compound of Claim 7 selected from:

2-mercapto-N-phenylbenzamide,

methyl 4-(2-mercaptobenzamido)benzoate,

methyl 4-(2-(methylthio)benzamido)benzoate,

methyl 4-(2-(S '-glutathionethio)benzamido)benzoate,

N-(5-(ethylthio)-l,3,4-thiadiazol-2-yl)-2-mercaptobenzamide.

10. The composition of Claims 1 and 7 comprising a pharmaceutically acceptable carrier selected from a diluent, binder, lubricant, disintegrator, filler, pH modifying agent, preservative, antioxidant, solubility enhancer, or coating.

11. A pharmaceutical composition comprising a compound as in Claims 1 -9 further comprising a second active ingredient. Emory Ref.: 10004 PCT

12. A method of treating a patient suffering from a Nox-related disease or disorder comprising administering to a patient in need thereof the composition of Claims 1 or 7.

13. The method of Claim 12, wherein the disease or disorder is selected from the group consisting of hypertension, chronic obstructive pulmonary disease (COPD), Alzheimer's disease (AD), Parkinson's disease (PD), acute respiratory distress syndrome (ARDS), acute lung injury (ALI), amyotrophic lateral sclerosis (ALS), atherosclerosis, aging-related deafness, inflammatory diseases, such as arthritis; various cancers such as colon cancer, prostate cancer, fibrotic diseases, such as liver fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic systemic fibrosis, Crohn's disease, and

14. A method of preventing ischemic injury comprising administering to a subject before, during or after an organ transplant a composition of Claims 1 o 7.

15. A storage medium comprising a compound as in Claims 1 or 7 and a salt, an amino acid, saccharide, polysaccharide, vitamin, or adenosine triphosphate.

16. A cosmetic composition comprising a compound of formula I or II as provide in Claims 1 or 7.