ANTI-INFLAMMATORY AGENTS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 61/706,329, filed September 27, 2012, and to U.S. Provisional Application No. 61/808,487, filed April 4, 2013, the contents of each of which are herein incorporated by reference.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under contract no. R01-CA- 121107, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present disclosure relates generally to salicylate-based compounds and compositions, methods of using the compounds and compositions, and processes for preparing the compounds and compositions.

BACKGROUND

[0004] Aspirin is a well established drug belonging to the class of non steroidal antiinflammatory drugs (NSAIDs) which displays a variety of actions including antiinflammatory, analgesic, antipyretic and antithrombotic activities. The major drawback which limits its use is a relevant gastrotoxicity that is responsible for gastric ulceration, exacerbation of peptic ulcer symptoms, gastrointestinal hemorrhage and erosive gastritis. Aspirin prodrugs, such as NCX-4016, have been designed to mitigate the gastrotoxicity. However, despite these efforts, there remains a need for improved salicylate-based compounds.

SUMMARY

[0005] In one aspect, disclosed is a compound of formula (I),

(I)

or a pharmaceutically acceptable salt thereof,

wherein,

Xi is selected from the group consisting of O and S;

Ari is selected from the group consisting of a bond, aryl and arylalkyl;

Gi is selected from the group consisting of a bond and alkylenyl;

X ₂ is selected from the group consisting of a bond, O and S;

Y is selected from the group consisting of a bond, sulfonyl and carbonyl;

Z is selected from the group consisting of alkyl, aryl, halogen, hydroxyalkyl, nitro and alkenylenyl-Ri;

Ri is alkoxycarbonyl or -Y'-X ₂ '-Gi'-Ari'-Xi'-arylcarbonyl;

Y' is selected from the group consisting of a bond, sulfonyl and carbonyl;

X ₂ ' is selected from the group consisting of a bond, O and S;

Gi' is selected from the group consisting of a bond and alkylenyl;

Ari' is selected from the group consisting of a bond, aryl and arylalkyl; and

Xi' is selected from the group consisting of O and S.

[0006] In certain embodiments, Ari is phenyl.

[0007] In certain embodiments, Ari is naphthyl.

[0008] In certain embodiments, Gi is methylenyl.

[0009] In certain embodiments, Gi is ethylenyl.

[0010] In certain embodiments, Z is aryl.

[0011] In certain embodiments, Z is alkenylenyl-Ri.

[0012] In certain embodiment, Ri is alkoxycarbonyl.

[0013] In certain embodiments, Ri is formula (i)

(i).

[0014] In certain embodiments, Ari is phenyl; and Gi is methylenyl.

[0015] In certain embodiments, Ari is phenyl; Gi is methylenyl; and X2 is O.

[0016] In certain embodiments, Ari is phenyl; Gi is methylenyl; X ₂ is O; and Y is sulfonyl.

[0017] In certain embodiments, Ari is phenyl; Gi is methylenyl; X ₂ is O; and Y is carbonyl.

[0018] In certain embodiments, the compound of formula (I) is selected from the group consisting of:

3-((tosyloxy)methyl)phenyl 2-acetoxybenzoate;

3-((((4-fluorophenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

3-((((4-acetylphenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

3-((2-acetoxybenzoyl)oxy)benzyl 2-acetoxybenzoate;

3- ((((4-chlorophenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

4- ((((4-fluorophenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

4-((2-acetoxybenzoyl)oxy)benzyl 2-acetoxybenzoate;

4-((2-acetoxybenzoyl)oxy)benzyl ethyl maleate;

4-((2-acetoxybenzoyl)oxy)benzyl ethyl fumarate;

4-(2-bromoethyl)phenyl 2-acetoxybenzoate;

4-(2-(tosyloxy)ethyl)phenyl 2-acetoxybenzoate;

4-(2-((methylsulfonyl)oxy)ethyl)phenyl 2-acetoxybenzoate;

3- ((((4-isopropylphenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

4- ((((4-acetylphenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

4-(((2-hydroxyethyl)thio)methyl)phenyl 2-acetoxybenzoate;

2-(((2-(nitrooxy)ethyl)thio)carbonyl)phenyl acetate;

4-(2,4-dinitrophenoxy)benzyl 2-acetoxybenzoate; 4-(((2-acetoxybenzoyl)oxy)methyl)naphthalen- 1 -yl 2-acetoxybenzoate;

2-(((4-(bromomethyl)phenyl)thio)carbonyl)phenyl acetate;

2-(((4-((nitrooxy)methyl)phenyl)thio)carbonyl)phenyl acetate;

4-((2-acetoxybenzoyl)thio)benzyl 2-acetoxybenzoate;

2- (((4-(((2-hydroxyethyl)thio)methyl)phenyl)thio)carbonyl)phen yl acetate;

4-(bromomethyl)naphthalen- 1 -yl 2-acetoxybenzoate;

4-((nitrooxy)methyl)naphthalen- 1 -yl 2-acetoxybenzoate;

4-((2-acetoxybenzoyl)thio)benzyl acetate;

3- (azidomethyl)-4-((nitrooxy)methyl)phenyl 2-acetoxybenzoate;

3- (azidomethyl)-4-(bromomethyl)phenyl 2-acetoxybenzoate;

4- ((2-acetoxybenzoyl)oxy)-2-(azidomethyl)benzyl 2-acetoxybenzoate;

4-(acetoxymethyl)-3-(azidomethyl)phenyl 2-acetoxybenzoate;

bis(4-((2-acetoxybenzoyl)oxy)benzyl) fumarate;

2-((4-nitrophenyl)sulfonyl)ethyl 2-acetoxybenzoate;

4-((2-(ethoxycarbonyl)phenoxy)methyl)phenyl 2-acetoxybenzoate; and

2-((2-(ethoxycarbonyl)phenoxy)methyl)phenyl 2-acetoxybenzoate,

or a pharmaceutically acceptable salt thereof.

[0019] In certain embodiments, a compound of the invention is selected from the group consisting of:

1- (4-(bromomethyl)phenoxy)-2,4-dinitrobenzene;

4-(2,4-dinitrophenoxy)benzyl nitrate;

2- (ethoxycarbonyl)phenyl ethyl fumarate; and

bis(2-(ethoxycarbonyl)phenyl) fumarate,

or a pharmaceutically acceptable salt thereof.

[0020] In certain embodiments, a compound of the invention is used for modulating Nrf2 induction. In certain embodiments, a compound of the invention is used for inhibiting NFKB. In certain embodiments, a compound of the invention is used for treating or preventing inflammation. In certain embodiments, a compound of the invention is used for treating or preventing cancer. In certain embodiments, a compound of the invention is used for treating or preventing breast cancer, colon cancer, or colorectal cancer.

[0021] In another aspect, disclosed is a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention (e.g., formula (I)) in combination with a pharmaceutically suitable carrier.

[0022] In another aspect, disclosed is a method of modulating the activity of Nrf2, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the invention.

[0023] In another aspect, disclosed is a method of inhibiting NFKB, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the invention.

[0024] In another aspect, disclosed is a method of inhibiting the growth of cancer cells, comprising contacting said cells with an effective amount of a compound of the invention.

[0025] In another aspect, disclosed is a method for treating or preventing inflammation, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the invention.

[0026] In another aspect, disclosed is a method for treating or preventing cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the invention. In certain embodiments, the cancer is breast cancer, colon cancer, or colorectal cancer.

[0027] The compounds, compositions, methods and processes are further described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows that GTCpFE inhibits TNFa-induced NFkB-RE activity in MCF-7 breast cancer cells.

[0029] FIG. 2 shows that GTCpFE inhibits TNFa-induced NFkB target genes in MCF-7 breast cancer cells.

[0030] FIG. 3 shows that GTCpFE inhibits TNFa-induced NFkB target genes in MDA- MB-231.

[0031] FIG. 4 shows that GTCpFE inhibits TNFa-induced NFkB target genes in BT474 breast cancer cells. [0032] FIG. 5 shows that GTCpFE and GTCmFE inhibit TNFa-induced NFkB-RE activity in breast cancer cells.

[0033] FIGS. 6A-6C show that GTCpFE and GTCmFE inhibit TNFa-induced NFkB target gene expression in breast cancer cells.

[0034] FIG. 7 shows that GTCpFE inhibits p65 DNA binding in MCF-7 mammospheres.

[0035] FIGS. 8A-8C show that GTCpFE inhibits NFkB target genes in MCF-7 mammospheres.

[0036] FIGS. 9A-9C show dose response curves for the effect of DMF and GTCpFE on mammosphere formation.

[0037] FIG. 10 shows that GTCpFE reduces the population of CD44+ CD24- MDA-MB- 231 cells.

[0038] FIG. 1 1 shows the effect of DMF and GTCpFE on nuclear and phospho p65 and p50 in mammospheres.

[0039] FIG. 12 shows that both p(ASA) ₂ and o(ASA) ₂ show high efficacy towards activation of Nrf2/ARE signaling.

DETAILED DESCRIPTION

[0040] Disclosed herein are anti-inflammatory, salicylate-based compounds. In certain embodiments, the disclosed compounds incorporate sulfonate leaving groups designed to control spatiotemporal release of anti-inflammatory components and control electronic (Hammett) and lipophilicity (Hansch, tPSA, and ClogP) parameters. In certain embodiments, the disclosed compounds incorporate carboxylate leaving groups, including fumarate, maleate, and salicylate. These leaving groups are pharmaceutically acceptable anions and can contribute to electrophile-induced activity. In certain embodiments, the disclosed compounds contain a thioester susceptible to bioactivation. In certain embodiments, the disclosed compounds exploit bioactivation by glutathione-S-transferase that is upregulated in cancer cells. These agents may include a leaving group that is for example, sulfonate or nitrate.

[0041] The compounds may be used for cancer chemoprevention and therapy. The modulated cytotoxic, genotoxic, antiproliferative, cytoprotective, and apoptotic actions of the disclosed compounds may be attributed to bioactivation to an electrophile (e.g., a quinone- methide electrophile). In addition to anti-inflammatory actions of the compounds, the quinone-methide electrophile itself may provide induction of phase 2 enzymes, and induction of anti-inflammatory mechanisms, including inhibition of NFKB signaling. The disclosed compounds may provide minimal gastrotoxicity, a serious side effect with aspirin and other NSAIDs.

1. Definition of Terms

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0043] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments "comprising," "consisting of and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

[0044] As used herein, the term "suitable substituent" is intended to mean a chemically and pharmaceutically acceptable functional group i.e., a moiety that does not negate the biological activity of the inventive compounds. Such suitable substituents may be routinely selected by those skilled in the art. Illustrative examples of suitable substituents include, but are not limited to halo groups, perfluoroalkyl groups, perfluoroalkoxy groups, alkyl groups, alkenyl groups, alkynyl groups, hydroxy groups, halo groups, oxo groups, mercapto groups, alkylthio groups, alkoxy groups, nitro groups, azidealkyl groups, aryl or heteroaryl groups, aryloxy or heteroaryloxy groups, aralkyl or heteroaralkyl groups, aralkoxy or heteroaralkoxy groups, HO— (C=0)— groups, heterocylic groups, cycloalkyl groups, amino groups, alkyl - and dialkylamino groups, carbamoyl groups, alkylcarbonyl groups, alkylcarbonyloxy groups, alkoxycarbonyl groups, alkylaminocarbonyl groups, dialkylamino carbonyl groups, arylcarbonyl groups, aryloxycarbonyl groups, alkylsulfonyl groups, arylsulfonyl groups and the like. Those skilled in the art will appreciate that many substituents can be substituted by additional substituents. [0045] As used herein, the term "alkenyl" refers a straight or branched hydrocarbon chain containing from 2 to 10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5- hexenyl, 2-heptenyl, 2-methyl-l-heptenyl, and 3-decenyl. Alkenyl groups of the present invention may be unsubstituted or substituted by one or more suitable substituents, preferably 1 to 3 suitable substituents, as defined above.

[0046] As used herein, the term "alkenylenyl" refers to a divalent group derived from a straight or branched chain containing from 2 to 10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. Representative examples of alkenylenyl include, but are not limited to, cis-ethylenyl and trans-ethylenyl.

[0047] As used herein, the term "alkoxy" refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert- butoxy, pentyloxy, and hexyloxy.

[0048] As used herein, the term "alkoxyalkoxy" refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through another alkoxy group, as defined herein. Representative examples of alkoxyalkoxy include, but are not limited to, tert- butoxymethoxy, 2-ethoxyethoxy, 2 -methoxy ethoxy, and methoxymethoxy.

[0049] As used herein, the term "alkoxyalkyl" refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of alkoxyalkyl include, but are not limited to, tert-butoxymethyl, 2- ethoxyethyl, 2 -methoxy ethyl, and methoxymethyl.

[0050] As used herein, the term "alkoxycarbonyl" refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein. Representative examples of alkoxycarbonyl include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, and tert-butoxycarbonyl.

[0051] As used herein, the term "alkyl" refers to a linear or branched hydrocarbon radical having the specified number of carbon atoms. The term "Ci-C6-alkyl" is defined to include alkyl groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement. For example, "Ci-C6-alkyl" specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl, t- butyl, i-butyl, pentyl, and hexyl. Alkyl groups of the present invention may be unsubstituted or substituted by one or more suitable substituents, preferably 1 to 3 suitable substituents, as defined above. [0052] As used herein, the term "alkylenyl" refers to a divalent group derived from a linear or branched chain containing from 1 to 10 carbon atoms. Representative examples of alkylenyl include, but are not limited to, methylenyl and ethylenyl.

[0053] As used herein, the term "alkylamino" refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an amino group, as defined herein.

Representative examples of alkylamino include, but are not limited to, methylamino, ethylamino, and sec-butylamino.

[0054] As used herein, the term "alkylaminoalkyl" refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an aminoalkyl group, as defined herein. Representative examples of alkylaminoalkyl groups include, but are not limited to, methylaminoethyl and methylamino-2-propyl.

[0055] As used herein, the term "alkylcarbonyl" refers to an alkyl group appended to the parent molecular moiety through a carbonyl group, as defined herein. Representative examples of alkylcarbonyl include, but are not limited to, acetyl, 1-oxopropyl, 2,2-dimethyl-

1- oxopropyl, 1-oxobutyl, and 1-oxopentyl.

[0056] As used herein, the term "alkylcarbonyloxy" refers to an alkylcarbonyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.

Representative examples of alkylcarbonyloxy include, but are not limited to, acetyloxy, ethylcarbonyloxy, and tert-butylcarbonyloxy.

[0057] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon radical having 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons, and having one or more carbon-carbon triple bonds. Alkynyl groups of the present invention include, but are not limited to, ethynyl, propynyl, and butynyl. Alkynyl groups of the present invention may be unsubstituted or substituted by one or more suitable substituents, preferably 1 to 3 suitable substituents, as defined above.

[0058] As used herein, the term "amino" refers to an -NH ₂ group.

[0059] As used herein, the term "aminoalkyl" refers to at least one amino group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of aminoalkyl include, but are not limited to, aminomethyl,

2- aminoethyl, and 2-aminopropyl.

[0060] As used herein, the term "aryl" means monocyclic, bicyclic, or tricyclic aromatic radicals. Representative examples of the aryl groups include, but are not limited to, phenyl, dihydroindenyl, indenyl, naphthyl, dihydronaphthalenyl, and tetrahydronaphthalenyl. Aryl groups of the present invention may be optionally substituted by one or more suitable substituents, preferably 1 to 5 suitable substituents, as defined above.

[0061] As used herein, the term "arylalkyl" refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

Representative examples of arylalkyl include, but are not limited to, phenylmethyl and phenylethyl.

[0062] As used herein, the term "arylcarbonyl" refers to an aryl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein.

[0063] As use herein, the term "azide" refers to an -N=N ⁺ =N ^" (-N ₃ ) group.

[0064] As used herein, the term "azidealkyl" refers to an azide group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

Representative examples of azidealkyl include, but are not limited to, azidemethyl and azideethyl.

[0065] As used herein, the term "carbonyl" or "(C=0)" (as used in phrases such as alkylcarbonyl, alkyl -(C=0)— or alkoxycarbonyl) refers to the joinder of the >C=0 moiety to a second moiety such as an alkyl or amino group (i.e. an amido group).

Alkoxycarbonylamino (i.e. alkoxy(C=0)— NH— ) refers to an alkyl carbamate group. The carbonyl group is also equivalently defined herein as (C=0). Alkylcarbonylamino refers to groups such as acetamide.

[0066] As used herein, the term "cycloalkyl" refers to a mono, bicyclic or tricyclic carbocyclic radical (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclopentenyl, cyclohexenyl, bicyclo[2.2.1]heptanyl,

bicyclo[3.2.1]octanyl and bicyclo[5.2.0]nonanyl, etc.); optionally containing 1 or 2 double bonds. Cycloalkyl groups of the present invention may be unsubstituted or substituted by one or more suitable substituents, preferably 1 to 5 suitable substituents, as defined above.

[0067] As used herein, the term "di(alkyl)amino" refers to two independently selected alkyl groups, as defined herein, appended to the parent molecular moiety through an amino group, as defined herein. Representative examples of di(alkyl)amino include, but are not limited to, Ν,Ν-dimethylamino, N-ethyl-N-methylamino, and N-isopropyl-N-methylamino.

[0068] As used herein, the term "di(alkyl)aminoalkyl" refers to a di(alkyl)amino group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of di(alkyl)aminoalkyl include, but are not limited to, N,N- dimethylaminoethyl and N,N-methyl(2-propyl)aminoethyl. [0069] As used herein, the term "halogen" or "halo" refers to a fluoro, chloro, bromo or iodo radical.

[0070] As used herein, the term "haloalkoxy" refers to an alkoxy group, as defined herein, substituted by one, two, three, or four halogen atoms. Representative examples of haloalkoxy include, but are not limited to, chloromethoxy, 2-fluoroethoxy, trifluoromethoxy, and pentafluoroethoxy.

[0071] As used herein, the term "haloalkyl" refers to an alkyl group, as defined herein, substituted by one, two, three, or four halogen atoms. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and 4,4,4,-trifluorobutyl.

[0072] As used herein, the term "heteroaryl" refers to a monocyclic heteroaryl or a bicyclic heteroaryl. The monocyclic heteroaryl is a five- or six-membered ring. The five- membered ring contains two double bonds. The five-membered ring may contain one heteroatom selected from O or S; or one, two, three, or four nitrogen atoms and optionally one oxygen or sulfur atom. The six-membered ring contains three double bonds and one, two, three or four nitrogen atoms. Representative examples of monocyclic heteroaryl include, but are not limited to, furanyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, 1,3- oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, 1,3-thiazolyl, thienyl, triazolyl, and triazinyl. The bicyclic heteroaryl includes a monocyclic heteroaryl fused to a phenyl, or a monocyclic heteroaryl fused to a monocyclic cycloalkyl, or a monocyclic heteroaryl fused to a monocyclic cycloalkenyl, or a monocyclic heteroaryl fused to a monocyclic heteroaryl, or a monocyclic heteroaryl fused to a monocyclic heterocycle. Representative examples of bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzothienyl, benzoxazolyl, benzimidazolyl,

benzoxadiazolyl, 6,7-dihydro-l,3-benzothiazolyl, imidazo[l,2-a]pyridinyl, indazolyl, indolyl, isoindolyl, isoquinolinyl, naphthyridinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, thiazolo[5,4-b]pyridin-2-yl, thiazolo[5,4-d]pyrimidin-2-yl, and 5,6,7,8-tetrahydroquinolin-5- yl. Heteroaryl groups of the present invention may be unsubstituted or substituted by one or more suitable substituents, preferably 1 to 5 suitable substituents, as defined above.

[0073] As used herein, the term "heterocycle" or "heterocyclyl" refers to a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. The monocyclic heterocycle is a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from the group consisting of oxygen, nitrogen, phosphorus and sulfur. The three- or four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of oxygen, nitrogen, phosphorus and sulfur. The five- membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus and sulfur. The six-membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus and sulfur. The seven- and eight- membered rings contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus and sulfur. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3- dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, phosphinane, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,

tetrahydropyrimidinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1, 1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a bridged monocyclic heterocycle ring system in which two non- adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3- dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, azabicyclo[2.2.1]heptyl (including 2- azabicyclo[2.2. l]hept-2-yl), 2,3-dihydro-lH-indolyl, isoindolinyl,

octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, 9-phosphabicyclo[3.3. l]nonane, 8-phosphabicyclo[3.2.1]octane, and tetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a phenyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro- 1 H- 1 ,4- methanocyclopenta[c]furan, aza-admantane (l-azatricyclo[3.3.1.1 ³ ' ⁷ ]decane), oxa- adamantane (2-oxatricyclo[3.3.1.1 ³ ' ⁷ ]decane), and 2,4,6-trioxa-8- phosphatricyclo[3.3.1.13,7]decane. Heterocyclic groups of the present invention may be unsubstituted or substituted by one or more suitable substituents, preferably 1 to 3 suitable substituents, as defined above.

[0074] As used herein, the term "hydroxy" refers to an -OH group.

[0075] As used herein, the term "hydroxyalkoxy" refers to an alkoxy group, as defined herein, substituted by at least one hydroxy group. Representative examples of hydroxyalkoxy include, but are not limited to, hydroxyethoxy, and 2-hydroxypropoxy.

[0076] As used herein, the term "hydroxyalkyl" refers to an alkyl group, as defined herein, substituted by at least one hydroxy group. Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2,3- dihydroxypropyl, 2,3-dihydroxypentyl, 4-hydroxybutyl, 2-ethyl-4-hydroxyheptyl, 3,4- dihydroxybutyl, and 5-hydroxypentyl.

[0077] As used herein, the term "methylenedioxy" refers to a— OCH ₂ 0— group wherein the oxygen atoms of the methylenedioxy are attached to the parent molecular moiety through two adjacent carbon atoms.

[0078] As used herein, the term "nitrogen protecting group" refers to groups intended to protect an amino group against undesirable reactions during synthetic procedures.

Representative nitrogen protecting groups include acetyl, benzoyl, benzyl,

benzyloxycarbonyl (Cbz), formyl, phenylsulfonyl, tert-butoxycarbonyl (Boc), tert- butylacetyl, trifluoroacetyl, and triphenylmethyl (trityl).

[0079] As used herein, the term "oxo" refers to a double bonded oxygen (=0) radical wherein the bond partner is a carbon atom. Such a radical can also be thought as a carbonyl group.

[0080] As used herein, the term "sulfonyl" refers to an >S(0)2 group.

[0081] A prefix attached to a multi-component substituent only applies to the first component it precedes. To illustrate, the term "alkylcycloalkyl" contains two components: alkyl and cycloalkyl. Thus, the Ci-C6-prefix on Ci-C6-alkylcycloalkyl means that the alkyl component of the alkylcycloalkyl contains from 1 to 6 carbon atoms; the Ci-C6-prefix does not describe the cycloalkyl component. To illustrate further, the prefix "halo" on

haloalkoxyalkyl indicates that only the alkoxy component of the alkoxyalkyl substituent is substituted with one or more halogen radicals. If the halogen substitution may only occur on the alkyl component, the substituent would instead be described as "alkoxyhaloalkyl." [0082] A substituent is "substitutable" if it comprises at least one carbon or nitrogen atom that is bonded to one or more hydrogen atoms. Thus, for example, hydrogen, halogen, and cyano do not fall within this definition. In addition, a sulfur atom in a heterocyclyl containing such atom is substitutable with one or two oxo substituents.

[0083] If a substituent is described as being "substituted", a non-hydrogen radical is in the place of hydrogen radical on a carbon or nitrogen of the substituent. Thus, for example, a substituted alkyl substituent is an alkyl substituent in which at least one non-hydrogen radical is in the place of a hydrogen radical on the alkyl substituent. To illustrate, monofluoroalkyl is alkyl substituted with a fluoro radical, and difluoroalkyl is alkyl substituted with two fluoro radicals. It should be recognized that if there is more than one substitution on a substituent, each non-hydrogen radical may be identical or different (unless otherwise stated).

[0084] When a substituent is referred to as "unsubstituted" or not referred to as

"substituted" or "optionally substituted", it means that the substituent does not have any substituents. If a substituent is described as being "optionally substituted", the substituent may be either (1) not substituted or (2) substituted. If a substituent is described as being optionally substituted with up to a particular number of non-hydrogen radicals, that substituent may be either (1) not substituted; or (2) substituted by up to that particular number of non-hydrogen radicals or by up to the maximum number of substitutable positions on the substituent, whichever is less. Thus, for example, if a substituent is described as a heteroaryl optionally substituted with up to 3 non-hydrogen radicals, then any heteroaryl with less than 3 substitutable positions would be optionally substituted by up to only as many non-hydrogen radicals as the heteroaryl has substitutable positions. To illustrate, tetrazolyl (which has only one substitutable position) would be optionally substituted with up to one non-hydrogen radical. To illustrate further, if an amino nitrogen is described as being optionally substituted with up to 2 non-hydrogen radicals, then a primary amino nitrogen will be optionally substituted with up to 2 non-hydrogen radicals, whereas a secondary amino nitrogen will be optionally substituted with up to only 1 non-hydrogen radical.

[0085] If substituents are described as being "independently selected" from a group, each substituent is selected independent of the other. Each substituent, therefore, may be identical to or different from the other substituent(s).

2. Compounds

[0086] Compounds of the invention include salicylate-based compounds.

[0087] In one aspect, compounds of the invention have formula (I),

(I),

or a pharmaceutically acceptable salt thereof,

wherein

Xi is selected from the group consisting of O and S;

Ari is selected from the group consisting of a bond, aryl and arylalkyl;

Gi is selected from the group consisting of a bond and alkylenyl;

X ₂ is selected from the group consisting of a bond, O and S;

Y is selected from the group consisting of a bond, sulfonyl and carbonyl;

Z is selected from the group consisting of alkyl, aryl, halogen, hydroxyalkyl, nitro and alkenylenyl-Ri;

Ri is alkoxycarbonyl or -Y'-X ₂ '-Gi'-Ari'-Xi'-arylcarbonyl;

Y' is selected from the group consisting of a bond, sulfonyl and carbonyl;

X ₂ ' is selected from the group consisting of a bond, O and S;

Gi' is selected from the group consisting of a bond and alkylenyl;

Ari' is selected from the group consisting of a bond, aryl and arylalkyl; and

Xi' is selected from the group consisting of O and S.

[0088] In certain embodiments, each aryl is independently unsubstituted or substituted with 1 or 2 substituents independently selected from the group consisting of alkyl, halogen, alkylcarbonyl, alkylcarbonyloxy, alkoxycarbonyl and nitro.

[0089] In certain embodiments, Xi is O.

[0090] In certain embodiments, Xi is S.

[0091] In certain embodiments, Ari is a bond.

[0092] In certain embodiments, Ari is aryl.

[0093] In certain embodiments, Ari is selected from the group consisting of phenyl and napthyl.

[0094] In certain embodiments, Ari is phenyl.

[0095] In certain embodiments, Ari is naphthyl. [0096] In certain embodiments, Ar is arylalkyl.

[0097] In certain embodiments, Ari is phenylmethyl.

[0098] In certain embodiments, Gi is a bond.

[0099] In certain embodiments, Gi is alkylenyl.

[00100] In certain embodiments, Gi is methylenyl.

[00101] In certain embodiments, Gi is ethylenyl.

[00102] In certain embodiments, X ₂ is a bond.

[00103] In certain embodiments, X ₂ is 0.

[00104] In certain embodiments, X ₂ is S.

[00105] In certain embodiments, Y is selected from the

carbonyl.

[00106] In certain embodiments Y is a bond.

[00107] In certain embodiments Y is sulfonyl.

[00108] In certain embodiments Y is carbonyl.

[00109] In certain embodiments Z is selected from the group consisting of alkyl and aryl.

[00110] In certain embodiments Z is selected from the group consisting of halogen, hydroxy alkyl and nitro.

[00111] In certain embodiments, Z is alkyl.

[00112] In certain embodiments, Z is aryl.

[00113] In certain embodiments, Z is halogen.

[00114] In certain embodiments Z is hydroxyalkyl.

[00115] In certain embodiments Z is nitro.

[00116] In certain embodiments, Z is alkenylenyl-Ri.

[00117] In certain embodiment, Ri is alkoxycarbonyl.

[00118] In certain embodiments Ri is -Y'-Xi'-Gi'-Ar

[00119] In certain embodiments Ri is formula (i)

(i).

[00120] In certain

[00121] In certain

[00122] In certain [00123] In certain embodiments, Ari is phenyl; Gi is alkylenyl; and X ₂ is O.

[00124] In certain embodiments, Ari is phenyl; Gi is alkylenyl; X ₂ is O; and Y is sulfonyl.

[00125] In certain embodiments, Ari is phenyl; Gi is alkylenyl; X ₂ is O; and Y is carbonyl.

[00126] In certain embodiments, Xi is O; Ari is phenyl; Gi is alkylenyl; X ₂ is O; Y is sulfonyl; and Z is aryl.

[00127] In certain embodiments, Xi is O; Ari is phenyl; Gi is alkylenyl; X ₂ is O; Y is carbonyl; and Z is aryl.

[00128] Exemplary compounds of the invention include, but are not limited to,

3-((tosyloxy)methyl)phenyl 2-acetoxybenzoate;

3-((((4-fluorophenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

3-((((4-acetylphenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

3-((2-acetoxybenzoyl)oxy)benzyl 2-acetoxybenzoate;

3- ((((4-chlorophenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

4- ((((4-fluorophenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

4-((2-acetoxybenzoyl)oxy)benzyl 2-acetoxybenzoate;

4-((2-acetoxybenzoyl)oxy)benzyl ethyl maleate;

4-((2-acetoxybenzoyl)oxy)benzyl ethyl fumarate;

4-(2-bromoethyl)phenyl 2-acetoxybenzoate;

4-(2-(tosyloxy)ethyl)phenyl 2-acetoxybenzoate;

4-(2-((methylsulfonyl)oxy)ethyl)phenyl 2-acetoxybenzoate;

3- ((((4-isopropylphenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

4- ((((4-acetylphenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate;

4-(((2-hydroxyethyl)thio)methyl)phenyl 2-acetoxybenzoate;

2-(((2-(nitrooxy)ethyl)thio)carbonyl)phenyl acetate;

l-(4-(bromomethyl)phenoxy)-2,4-dinitrobenzene;

4-(2,4-dinitrophenoxy)benzyl nitrate;

4-(2,4-dinitrophenoxy)benzyl 2-acetoxybenzoate;

4-(((2-acetoxybenzoyl)oxy)methyl)naphthalen- 1 -yl 2-acetoxybenzoate; 2-(((4-(bromomethyl)phenyl)thio)carbonyl)phenyl acetate;

2-(((4-((nitrooxy)methyl)phenyl)thio)carbonyl)phenyl acetate;

4-((2-acetoxybenzoyl)thio)benzyl 2-acetoxybenzoate;

2- (((4-(((2-hydroxyethyl)thio)methyl)phenyl)thio)carbonyl)phen yl acetate; 4-(bromomethyl)naphthalen- 1 -yl 2-acetoxybenzoate;

4-((nitrooxy)methyl)naphthalen- 1 -yl 2-acetoxybenzoate;

4-((2-acetoxybenzoyl)thio)benzyl acetate;

3- (azidomethyl)-4-((nitrooxy)methyl)phenyl 2-acetoxybenzoate;

3- (azidomethyl)-4-(bromomethyl)phenyl 2-acetoxybenzoate;

4- ((2-acetoxybenzoyl)oxy)-2-(azidomethyl)benzyl 2-acetoxybenzoate; 4-(acetoxymethyl)-3-(azidomethyl)phenyl 2-acetoxybenzoate;

2-(ethoxycarbonyl)phenyl ethyl fumarate;

bis(2-(ethoxycarbonyl)phenyl) fumarate;

bis(4-((2-acetoxybenzoyl)oxy)benzyl) fumarate;

2-((4-nitrophenyl)sulfonyl)ethyl 2-acetoxybenzoate;

4-((2-(ethoxycarbonyl)phenoxy)methyl)phenyl 2-acetoxybenzoate; and 2-((2-(ethoxycarbonyl)phenoxy)methyl)phenyl 2-acetoxybenzoate, or a pharmaceutically acceptable salt thereof.

In certain embodiments, compounds of the invention have formula (I-a)

(l-a) (l-b)

or a pharmaceutically acceptable salt thereof,

wherein Xi, Gi, X ₂ , Y and Z are as defined above;

R ² is G ₂ -N ₃ ;

G ₂ is alkylenyl; and m is 0 or 1.

[00130] In certain embodiments, compounds of the invention have formula (I-c),

or a pharmaceutically acceptable salt thereof,

wherein Xi, Y, Z, R ² and m are as defined above.

[00131] In certain embodiments, compounds of the invention have formula (I-d),

or a pharmaceutically acceptable salt thereof,

wherein Xi, Y, R ² and m are as defined above;

R ³ is selected from the group consisting of alkoxycarbonl, alkyl, alkylcarbonyl, alkylcarbonyloxy, halo and nitro; and

n is 0, 1 or 2.

[00132] In certain embodiments, compounds of the invention have formula (I-e) or (I-f),

(I-e) (I-f)

or a pharmaceutically acceptable salt thereof,

wherein Xi, Gi, X ₂ , Y, Z, R and m are as defined above.

[00133] In certain embodiments, compounds of the invention have formula (I-g),

or a pharmaceutically acceptable salt thereof, wherein Xi, Gi, X ₂ , Y, Z, R ² and m are as defined above.

[00134] Particularly preferred compounds of the invention include, but are not limited to,

4-((2-acetoxybenzoyl)oxy)benzyl ethyl fumarate;

2-(ethoxycarbonyl)phenyl ethyl fumarate; and

bis(2-(ethoxycarbonyl)phenyl) fumarate;

or a pharmaceutically acceptable salt thereof.

[00135] Compounds of the invention may contain asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Additional asymmetric centers may be present depending upon the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers and it is intended that all of the possible optical isomers and diastereomers in mixtures and as pure or partially purified compounds are included within the scope of this invention. The present invention is meant to comprehend all such isomeric forms of these compounds.

[00136] Compounds of the invention may be provided as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts are (a) acid addition salts organic and inorganic acids, for example, acid addition salts which may, for example, be hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, trifluoroacetic acid, formic acid and the like. Pharmaceutically acceptable salts can also be prepared from by treatment with inorganic bases, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Pharmaceutically acceptable salts can also be formed from elemental anions such as chlorine, bromine and iodine.

[00137] Compounds of the invention which contain basic nitrogen-containing groups can be quaternized using agents such as (Ci-C4)-alkyl halides, for example methyl, ethyl, isopropyl and tert-butyl chloride, bromide and iodide; di(Ci-C4)alkyl sulfates, for example dimethyl, diethyl and diamyl sulfate; (Cio-Ci ₈ )alkyl halides, for example decyl, do-decyl, lauryl, myristyl and stearyl chloride, bromide and iodide; and aryl-(Ci-C4)alkyl halides, for example benzyl chloride and phenethyl bromide. Both water- and oil-soluble compounds of the invention can be prepared using such salts. [00138] The acid-addition salts of basic compounds of the invention can be prepared by bringing the free base form into contact with a sufficient amount of the desired acid, causing the formation of the salt in a conventional manner. The free base can be regenerated by bringing the salt form into contact with a base and isolating the free base in a conventional manner.

[00139] The base-addition salts of acidic compounds of the invention can be prepared by bringing the free acid form into contact with a sufficient amount of the desired base, causing the formation of the salt in a conventional manner. The free acid can be regenerated by bringing the salt form into contact with an acid and isolating the free acid in a conventional manner.

[00140] If a compound of the invention contains more than one group which is capable of forming pharmaceutically acceptable salts, the compounds of the invention also encompasses multiple salts. Typical multiple salt forms include, for example, bitartrate, diacetate, difumarate, dimeglumine, di-phosphate, disodium and trihydrochloride.

[00141] Compounds of the invention can be prepared in the form of their hydrates. The term "hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate and the like.

[00142] Compounds of the invention can be prepared in the form of a solvate with any organic or inorganic solvent, for example alcohols such as methanol, ethanol, propanol and isopropanol, ketones such as acetone, aromatic solvents and the like.

[00143] Compounds of the invention can be prepared in any solid or liquid physical form. For example, the compound can be in a crystalline form, in amorphous form, and have any particle size. Furthermore, the compound particles may be micronized, or may be

agglomerated, particulate granules, powders, oils, oily suspensions or any other form of solid or liquid physical form.

[00144] Compounds of the invention may exhibit polymorphism. This invention further includes different polymorphs of the compounds of the present invention. The term

"polymorph" refers to a particular crystalline state of a substance, having particular physical properties such as X-ray diffraction, IR spectra, melting point, and the like.

3. Synthetic Methods

[00145] The compounds of the invention can be better understood in connection with the following synthetic schemes and methods which illustrate a means by which the compounds can be prepared. Scheme 1

GTc-pFE, GTc-pME, ASApASA, VL2- 45, VL-2-57, VL-2-58, VL-2-65

[00146] Compounds of the invention with a para-substitution pattern can be prepared as described in Scheme 1.

Scheme 2

GTc-mFE, ASAmASA, VL-2-64

[00147] Compounds of the invention with a meta-substitution pattern can be prepared as described in Scheme 2. Scheme 3

RG-1-96 RG-1-99

[00148] Compounds of the invention including 1 ,4-naphthalenes can be prepared as described in Scheme 3.

Scheme 4

Me Me Me

COOH SOCI ₂ r ^COOMe _B zCOCI/Et _3N i ^COOMe _{NBS Bz2} o ₂

^MeOH ^ HCT ^ BzO^^ CCI ₄ , reflux'

VL1-23, 93% VL1-28, 99%

VL1-30, 58% VL1-46, 77% VL1-47, 97%

[00149] Compounds of the invention, such as 3-azidomethyl-4-(2-acetoxybenzoyl)benzyl nitrate, can be prepared as described in Scheme 4.

Scheme 5

GTth-pBME (VAL-1-141) GTth-pBr (VAL-1-134) GTth-pNO (VAL-1-136)

[00150] Thio-containing compounds of the invention can be prepared as described in Scheme 5.

Scheme 6

ASA-CI VAL-1-114 VAL-1-117 GT-1031

[00151] A control compound of the invention can be prepared as described in Scheme 6.

Scheme 7

GT-DNP-001 (VAL-1 -123) GT-DNP-002 (VAL-1 -124)

[00152] Dinitrophenol (DNP) control compounds compounds of the invention can be prepared as described in Scheme 7.

[00153] In certain embodiments, the products may be further modified, for example, by manipulation of substituents. These manipulations may include, but are not limited to, reduction, oxidation, organometallic cross-coupling, alkylation, acylation, and hydrolysis reactions which are commonly known to those skilled in the art. In some cases, the order of carrying out the foregoing reaction schemes may be varied to facilitate the reaction or to avoid unwanted reaction products.

4. Pharmaceutical Compositions

[00154] The compounds of the invention may be incorporated into pharmaceutical compositions suitable for administration to a subject (such as a patient, which may be a human or non-human). Compounds of the invention may be provided in a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention in combination with a pharmaceutically suitable carrier. [00155] The pharmaceutical compositions may include a "therapeutically effective amount" or a "prophylactically effective amount" of the compounds. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.

Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

[00156] The pharmaceutical compositions may include pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier," as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[00157] The pharmaceutical compositions may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of composition that may be combined with a carrier material to produce a single dose vary depending upon the subject being treated, and the particular mode of administration.

[00158] Compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of a subject composition thereof as an active ingredient. Compositions of the present invention may also be administered as a bolus, electuary, or paste.

[00159] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the subject composition is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[00160] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. In certain embodiments, compounds of the invention may be formulated as a tablet, pill capsule or other appropriate ingestible formulation (collectively hereinafter "tablet"), to provide a therapeutic dose in 10 tablets or fewer. In another example, a therapeutic dose is provided in 50, 40, 30, 20, 15, 10, 5 or 3 tablets.

[00161] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the subject composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, cyclodextrins and mixtures thereof.

[00162] Suspensions, in addition to the subject composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[00163] Compositions for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room

temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent. Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[00164] Dosage forms for transdermal administration of a subject composition includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[00165] The ointments, pastes, creams and gels may contain, in addition to a subject composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. [00166] Powders and sprays may contain, in addition to a subject composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[00167] Compounds and compositions of the present invention may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension could be used. A drug delivery device for delivering aerosols may include a suitable aerosol canister with a metering valve containing a pharmaceutical aerosol formulation as described and an actuator housing adapted to hold the canister and allow for drug delivery. Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compounds contained in the subject compositions.

[00168] Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of a subject composition together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular subject composition, but typically include non-ionic surfactants (T weens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

[00169] Pharmaceutical compositions of this invention suitable for parenteral

administration comprise a compound of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[00170] Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate and cyclodextrins. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[00171] For application by the ophthalmic mucous membrane route, compositions of the present invention may be formulated as eyedrops or eye ointments. These formulations may be prepared by conventional means, and, if desired, the compositions may be mixed with any conventional additive, such as an excipient, a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent or a coating agent.

[00172] Methods of preparing these formulations include the step of bringing into association compositions of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association agents with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[00173] Compositions according to the invention can contain one or more additional agents, e.g., other antibiotics, anti-inflammatories, anti-fungals, steroids, decongestants, bronchodialators, and the like.

[00174] Diseases or disorders that may be treated or prevented by use of a pharmaceutical composition disclosed herein include, but are not limited to, pain, inflammation, arthritis, osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, headache, toothache, common cold, muscle ache, cardiovascular disease, cancer (e.g., colon cancer) or any combination thereof. In certain embodiments, pharmaceutical compositions disclosed herein may be administered to prevent or treat cardiovascular disease or cerebrovascular disease. In certain embodiments, pharmaceutical compositions as disclosed herein decrease the risk of a patient developing a gastric ulcer, duodenal ulcer, or both. In certain embodiments, a pharmaceutical composition disclosed herein may be administered to prevent or treat cancer, including but not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer;

choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; fibrosarcoma, gastric cancer; hepatoma, intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g., small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;

pancreatic cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; renal cancer, glioblastoma, adenocarcinoma, adenoma, astrocytoma, bladder tumor, bone carcinoma, brain carcinoma, Burkitt lymphoma, Kaposi Sarcoma, non-Hodgkins lymphoma, Hodgkins lymphoma, gastric tumor, breast carcinoma, cervical carcinoma, colon carcinoma, kidney carcinoma, liver carcinoma, lung carcinoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma, rectal carcinoma, skin carcinoma, stomach carcinoma, testis carcinoma, thyroid carcinoma, chondrosarcoma, choriocarcinoma, fibroma, fibrosarcoma, glioblastoma, glioma, hepatoma, histiocytoma, leiomyoblastoma,

leiomyosarcoma, leukemia, lymphoma, liposarcoma cell, mammary carcinoma,

medulloblastoma, melanoma, metastases, muscle tumor, myeloma, ovarian carcinoma, plasmacytoma, neuroblastoma, neuroglioma, osteogenic sarcoma, pancreatic tumor, pituitary carcinoma, renal tumors, retinoblastoma, rhabdomyosarcoma, sarcoma, testicular tumor, thymoma, uterine carcinoma, Wilms' tumor, as well as other carcinomas and sarcomas. In certain embodiments, the pharmaceutical compositions disclosed herein are administered to a patient to prevent or treat colon cancer, or colorectal cancer. In certain embodiments, the pharmaceutical compositions disclosed herein are administered to a patient to prevent or treat breast cancer.

5. Dosages and Dosing Schedules

[00175] The dosage regimen utilizing the compounds of the present invention can be selected in accordance with a variety of factors including type, species, age, weight, sex and the type of infection being treated; the severity (i.e., stage) of the disease to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to treat, for example, to prevent, inhibit (fully or partially) or arrest the progress of the disease.

[00176] The amount of the compound administered to a patient is less than an amount that would cause unmanageable toxicity in the patient. In the certain embodiments, the amount of the compound that is administered to the patient is less than the amount that causes a concentration of the compound in the patient's plasma to equal or exceed the toxic level of the compound. In one embodiment, the concentration of the compound in the patient's plasma is maintained at about 10 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 25 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 50 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 100 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 500 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 1000 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 2500 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 5000 nM. The optimal amount of the compound that should be administered to the patient in the practice of the present invention will depend on the particular compound used and the type of disease being treated.

[00177] In certain embodiments, the dosage of the subject compounds will generally be in the range of about 0.01 ng to about 10 g per kg body weight, specifically in the range of about 1 ng to about 0.1 g per kg, and more specifically in the range of about 100 ng to about 10 mg per kg.

[00178] For oral administration, suitable daily dosages are for example between about 2- 4000 mg administered orally once-daily, twice-daily or three times-daily, continuous (every day) or intermittently (e.g., 3-5 days a week). For example, when used to treat the desired disease, the dose of the compound can range between about 2 mg to about 2000 mg per day.

[00179] The compounds of the present invention may be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), and three times daily (TID). For administration once a day, a suitably prepared medicament would therefore contain all of the needed daily dose. For administration twice a day, a suitably prepared medicament would therefore contain half of the needed daily dose. For administration three times a day, a suitably prepared medicament would therefore contain one third of the needed daily dose.

[00180] In addition, the administration can be continuous, i.e., every day, or intermittently. The terms "intermittent" or "intermittently" as used herein means stopping and starting at either regular or irregular intervals.

[00181] Typically, an intravenous formulation may be prepared which contains a concentration of the compound of the invention of between about 1.0 mg/mL to about 10 mg/mL. In one example, a sufficient volume of intravenous formulation can be administered to a patient in a day such that the total dose for the day is between about 1 and about 1500 mg/mL.

[00182] Subcutaneous formulations, preferably prepared according to procedures well known in the art at a pH in the range between about 5 and about 12, also include suitable buffers and isotonicity agents. They can be formulated to deliver a daily dose of active compound in one or more daily subcutaneous administrations, e.g., one, two or three times each day.

[00183] The compounds can also be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime.

[00184] It should be apparent to a person skilled in the art that the various modes of administration, dosages and dosing schedules described herein merely set forth specific embodiments and should not be construed as limiting the broad scope of the invention. Any permutations, variations and combinations of the dosages and dosing schedules are included within the scope of the present invention.

6. Methods

[00185] A method of the invention may include administering a therapeutically effective amount of a compound or composition of the invention to a subject in need thereof. A method of the invention may include administering a prophylactically effective amount of a compound or composition of the invention to a subject in need thereof. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. A method of the invention may include a method of treating a disease or condition, a method of preventing a disease or condition, or a combination thereof.

[00186] Methods of treatment and/or prevention may include any number of modes of administering a compound or composition of the present invention. Modes of administration may include tablets, pills, dragees, hard and soft gel capsules, granules, pellets, aqueous, lipid, oily or other solutions, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, syrups, elixiers, solid emulsions, solid dispersions or dispersible powders.

Modulation of NrO Activity

[00187] Compounds of the invention can be used to modulate Nrf2 signaling (e.g., via covalent modification of Keapl). Transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) regulates a battery of genes encoding carcinogen-detoxifying enzymes and antioxidant proteins by binding to the antioxidant response element (ARE) promoter regulatory sequence. Under basal conditions, in which the redox homeostasis is maintained in cells, Nrf2 is sequestered in the cytoplasm by a protein known as Keapl, which targets Nrf2 for ubiquitination and degradation by the proteasome, and thus controls both the subcellular localization and steady-state levels of Nrf2. In response to oxidative stress or chemopreventive compounds, Keapl -mediated ubiquitination of Nrf2 is decreased significantly and the Nrf2 pathway is turned on. Thus, Keapl is a molecular switch that senses various stimuli and turns the Nrf2 pathway on and off. Administration of Nrf2- inducing agents has been shown to result in decreased carcinogenesis in animal models and altered carcinogen metabolism in humans. Clinical interventions have shown that Nrf2 inducers increase cytoprotective enzyme expression, resulting in modulation of aflatoxin disposition.

[00188] However, Nrf2 and its downstream genes are overexpressed in many cancer cell lines and human cancer tissues, giving cancer cells an advantage for survival and growth. Furthermore, Nrf2 is upregulated in resistant cancer cells and is thought to be responsible for acquired chemoresistance. Therefore, in certain embodiments, it may be necessary to inhibit the Nrf2 pathway, and thereby favor the action of concomitantly administered

chemotherapeutic agents.

[00189] Keapl contains the BTB domain mediating Keapl homodimer formation, the 'intervening region' (IVR) (amino acids 180-314), and the C-terminal Kelch domain that mediates binding to the Neh2 domain of Nrf2. Human Keapl contains 27 cysteine residues. Three key cysteine residues (C151, C273, and C288) have been identified. C151 is required for several Nrf2 inducers, such as sulforaphane (SFN) and tert-butylhydroquinone, to manifest their effect. Importantly, residues C273 and C288 at the IVR domain are necessary for Keapl to repress Nrf2. A single cysteine to serine mutation C273S or C288S render Keapl unable to repress Nrf2. The transgenic expression of mutant Keapl(C273A) and/or Keapl(C288A) protein in Keapl null mice failed to reverse constitutive Nrf2 activation, indicating that cysteine residues at positions 273 and 288 are essential for Keapl to repress Nrf2 activity in vivo. This suggests a critical role of these domains of Keapl in the regulation of the functional interaction of Keapl with rf2.

[00190] Compounds of the invention may modulate the interaction of Keapl with Nrf2 to prevent Keapl from binding to Nrf2, hence stimulating the activity of Nrf2, or they may modulate the interaction between Keapl and Cul3, and reduce Nrf2 degradation, hence stimulating the activity and/or expression level of Nrf2.

[00191] Alternatively, compounds of the invention may inhibit the activity of Nrf2. The compounds may prevent the release of Nrf2 from Keapl and/or stimulate its degradation. Multiple Keapl and Nrf2 mutations have been identified in cancers. These mutations effect the interaction of Keapl with Nrf2 by interfering with the repressive activity of Keapl toward Nrf2. Therefore, compounds of the invention may be useful for the treatment of conditions in which the repressive activity of Keapl towards Nrf2 is weakened or impaired due to mutations in Keapl, Nrf2, or both. [00192] Compounds of the invention may be used to treat or prevent Parkinson's disease, Parkinson's disease with dementia with Lewy body, multiple system atrophy (MSA), progressive supranuclear palsy (PSA), corticobasal degeneration (CBD), frontotemporal lobe degeneration, Huntington's disease, atherosclerosis, heart failure, myocardial infarction, Alzheimer's disease, Fragile X syndrome, and chronic fatigue. Compounds of the invention may provide a protective role (i.e. neuroprotective, cardioprotective, etc) by at least slowing, if not preventing the progress of neurodegenerative or cardiac diseases. Compounds of the invention may be used to treat or prevent cancer (e.g., liver cancer, lung cancer, breast cancer, prostate cancer, colon cancer, colorectal cancer, neuroblastoma, and leukemia).

[00193] In certain conditions, compounds of the invention may be used to treat cancers resistant to chemotherapeutic agents. The reduction in or the inability of Keapl of repressing Nrf2 (which may or may not be caused by mutations) results in increased Nrf2 activity. This increased activity over-stimulates the cellular Nrf2 -dependent response, and cause tumor cells to become resistant to chemotherapeutic drugs. Thus, tumor cells can use the Nrf2 pathway to their survival advantage, conferring resistance to chemotherapeutic agents. The compounds of the invention are useful for overcoming drug resistance in cancer

chemotherapies, as well as for inhibiting tumor growth, since tumor cells may depend on aberrant activation of Nrf2.

Modulation of NF B Activity

[00194] Compounds of the invention can be used to modulate NFKB activity. Nuclear factor-K B (NFKB) signaling is an essential signal transduction pathway involved in inflammatory responses, oncogenesis, viral infection, the regulation of cell proliferation and apoptosis and, in particularly in the case of B and T lymphocytes, in antigenic stimulation (Ghosh, 1998, Annu. Rev. Immunol, 16, 225-260; Karin, 1999, J. Biol. Chem., 274, 27339- 27342; Israel, 2000, Trends Cell. Biol, 10, 129-133; Santoro, 2003, EMBO J., 22, 2552- 2560). In mammalian cells, there are five NFKB family members that dimerize: RelA, RelB, c-Rel, NFKB 2/pl00/p52 and NFKB 1/ρ105/ρ50. NFKB, whose predominant form is a heterodimeric transcription factor composed of p50 and RelA subunits, remains sequestered in the cytoplasm through association with members of an inhibitory family of proteins known as IKB. Upon stimulation by the cytokines TNF-a and interleukin-1, endotoxin (LPS), microbial and viral infections, pro-inflammatory signals converge on the canonical IkB kinase complex (IKK), a protein complex that is composed of two kinase subunits,

ΙΚΚα/ΙΚΚ-l and ΙΚΚβ/ΙΚΚ-2 and a structural/regulatory subunit ΝΕΜΟ/ΙΚΚ-γ. Once activated IKK complex phosphorylates IkB proteins, triggering their ubiquitination and subsequent degradation by the proteasome. Free NFKB can then move into nucleus to initiate or up-regulate gene expression.

[00195] Although IKKa and ΙΚΚβ exhibit striking structural similarity (52%), genetic studies have shown that they are involved in two pathways for the activation of NFKB

(Pomerantz, 2002, Molecular Cell 2002 10: 693-695). ΙΚΚβ has been identified as the proinflammatory kinase responsible of activation of classical NFKB complexes, whereas IKKa in association with NFKB inducing kinase (NIK) plays an essential role in the non-canonical NFKB signaling pathway (Senftleben, 2001, 293: 1495-1499).

[00196] NFKB plays an essential role in the development and progression of cancer, including breast cancer. Animal studies suggest the presence of constitutively active NFKB at an early stage during neoplastic transformation of mammary cells (Clarkson et al, 2000, J Bio Chem. 275(17): 12737-42). NFKB inhibits apoptosis in mouse mammary epithelia (Sovak et al, 1999, Cell Growth Differ. 10(8):537-44) and selective activation of NFKB subunits have been found in human breast cancer cell lines and patient samples (Sovak et al, 1997, J Clin Invest. 100(12):2952-60; Cogswell et al, 2000, Oncogene 19(9): 1123-31). An inverse correlation between the levels of NFKB activation and estrogen receptor expression has been reported (Nakshatri et al, 1997, Mol Cell Biol. 17(7):3629-39) and inhibition of NFKB in breast cancer cells induces spontaneous apoptosis (Sovak et al, 1999, Cell Growth Differ. 10(8):537-44; Cogswell et al, 2000, Oncogene 19(9): 1123-31). Paclitaxel-induced sensitivity of breast cancer cell lines was enhanced by an NFKB inhibitor, parthenolide (Patel et al, 2000, Oncogene 19(36):4159-69; Newton et al, 1999, J Bio Chem. 274(26): 18827-35). The Mullerian inhibiting substance was also found to inhibit breast cancer growth through NFKB mediated pathway (Segev et al, 2000, J Bio Chem. 275(37):28371-9). Furthermore, the transactivation function of NFKB is negatively regulated by ΙκΒβΙ in breast cancer cell lines (Newton et al, 1999, J Bio Chem. 274(26): 18827-35). Lastly, overexpression of HER2/neu can activate NFKB through the activation of Akt pathway and block apoptosis (Zhou et al, 2000, J Bio Chem. 275(10:8027-31). All these reports together suggest that NFKB plays an important role in cancer generally and in breast cancer specifically.

[00197] Therefore, inhibition of NFKB activation represents a target for anti-inflammatory and anti-cancer drugs (Poulaki, 2002, Am J Pathol. 161 : 2229-2240). Among many protein actors in the NFKB signaling pathway, IKK complex represents one of the most attractive molecular targets for NFKB inhibitors. To minimize the potential toxicity effects in vivo, therapeutic success may greatly depend on the abilities of the NFKB inhibitors to block activating signals without modifying the basal level of NFKB activity. For example, May et al. described a cell-permeable peptidic inhibitor that specifically blocks the pro-inflammatory NFKB activation by disrupting the constitutive NEMO interaction with IKK kinases (May, 2000, Science 289, 1550-1554; May, 2002, J. Biol. Chem. 277, 45992-46000).

[00198] In addition to the above, Liao et al. (N. Engl. J. Med. 2012; 367: 1596-160) recently indicated that aspirin/salicyate therapy increases the 5-year survival rate from 76% to 97% of cancer patients having a mutation at the PIK3CA gene with the strongest effect of aspirin use in patients who had tumors with both a PIK3CA mutation and PTGS2 expression.

Experimental observation that aspirin can induce cell apoptosis through PTGS2-independent pathways coupled with information about observed cross talk between the two pathways supports the use of salicylate-based therapy in subtypes of colorectal cancer.

[00199] Accordingly, compounds of the invention may be used, in an effective amount, for the treatment or prevention of conditions including, but not limited to, cancer (e.g., breast cancer, colon cancer, colorectal cancer), tumorogenesis, and inflammatory conditions including, but not limited to, type I hypersensitivity, atopy, anaphylaxis, asthma, osteoarthritis, rheumatoid arthritis, septic arthritis, gout, juvenile idiopathic arthritis, still's disease, ankylosing spondylitis, inflammatory bowel disease, Crohn's disease or

inflammation associated with vertebral disc herniation. Compounds of the invention may be directed to the treatment of diseases related to dysfunction of cell proliferation, the immune system and/or inflammation. Compounds of the invention may be administered to prevent or treat cancer, including but not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; fibrosarcoma, gastric cancer; hepatoma, intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g., small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; renal cancer, glioblastoma, adenocarcinoma, adenoma, astrocytoma, bladder tumor, bone carcinoma, brain carcinoma, Burkitt lymphoma, Kaposi Sarcoma, non-Hodgkins lymphoma, Hodgkins lymphoma, gastric tumor, breast carcinoma, cervical carcinoma, colon carcinoma, kidney carcinoma, liver carcinoma, lung carcinoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma, rectal carcinoma, skin carcinoma, stomach carcinoma, testis carcinoma, thyroid carcinoma, chondrosarcoma, choriocarcinoma, fibroma, fibrosarcoma, glioblastoma, glioma, hepatoma, histiocytoma, leiomyoblastoma, leiomyosarcoma, leukemia, lymphoma, liposarcoma cell, mammary carcinoma, medulloblastoma, melanoma, metastases, muscle tumor, myeloma, ovarian carcinoma, plasmacytoma, neuroblastoma, neuroglioma, osteogenic sarcoma, pancreatic tumor, pituitary carcinoma, renal tumors, retinoblastoma, rhabdomyosarcoma, sarcoma, testicular tumor, thymoma, uterine carcinoma, Wilms' tumor, as well as other carcinomas and sarcomas. In certain embodiments, the pharmaceutical compositions disclosed herein are administered to a patient to prevent or treat colon cancer, or colorectal cancer. In certain embodiments, the compounds of the invention are administered to a patient to prevent or treat breast cancer.

[00200] Also disclosed herein are methods of manufacturing medicaments for the treatment or prevention disease and conditions. In certain embodiments, disclosed is a method of manufacturing a medicament for the treatment or prevention of inflammation using a compound of the invention. In certain embodiments, disclosed is a method of manufacturing a medicament for the treatment or prevention of cancer (e.g., colon cancer, breast cancer) using a compound of the invention.

7. Kits

[00201] This invention also provides kits for conveniently and effectively implementing the methods of this invention. Such kits may include a compound or composition of the invention, and optionally one or more of instructions, packaging, and dispensers. Kit components may be packaged for either manual or partially or wholly automated practice of the foregoing methods. In other embodiments involving kits, this invention contemplates a kit including compositions of the present invention, and optionally instructions for their use.

8. Examples

[00202] The foregoing may be better understood by reference to the following examples, which are presented for purposes of illustration and are not intended to limit the scope of the invention.

Preparation of Compounds

[00203] ^l H nuclear magnetic resonance (NMR) and ¹³ C NMR spectra were determined using 400 MHz FT NMR spectrometers. The chemical shifts (δ) are expressed in parts per million (ppm) relative to tetramethylsilane (TMS) as the internal standard. Splitting patterns are as follows: s, single; d, doublet; br, broad; m, multiplet. All mass spectra were recorded with Shimadzu IT-TOF spectrometer. Chemicals were purchased from Sigma Aldrich. General Procedure

[00204] Under a positive pressure of argon and free of moisture, a solution of either 3- or 4- hydroxymethylphenol ester of 2-acetyloxybenzoic acid (100 mg, 0.349 mmol), 4- dimethylaminopyridine (4 mg, 0.033 mmol) and triethylamine (53 mg, 0.523 mmol) in anhydrous tetrahydrofuran (5 mL) was cooled to 0 °C, and a solution of an appropriate electrophile (0.419 mmol) in anhydrous tetrahydrofuran (5 mL) was added dropwise over a period of 10 min. The resulting solution was stirred at 0-5 °C for 2-4 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and the diluted solution was washed with brine (50 mL x 3), dried over sodium sulfate, evaporated to dryness, and the residue was purified by column chromatography on silica gel.

Example 1

3-((tosyloxy)methyl)phenyl 2-acetoxybenzoate

-393)

[00205] (11.80 mg, >97% pure). ¾ NMR (CDC1 ₃ ): δ 2.30 (s, 3 H, CH ₃ ); 2.43 (s, 3 H, CH ₃ ); 5.07 (s, 2H, OCH ₂ ); 6.96-7.00 (m, 1 H, Ar); 7.06-7.19 (m, 3 H, Ar); 7.30-7.45 (m, 4 H, Ar); 7.64-7.70 (m, 1 H, Ar); 7.68-7.70 (d, 2 H, J = 10.8 Hz, Ar); 8.22 (dd, 1 H, J = 12.0, 1.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.62, 162.70, 151.19, 150.61, 145.02, 135.19, 134.79, 132.95, 132.11, 129.94, 129.87, 127.94, 126.22, 125.92, 124.07, 122.33, 122.25, 121.57, 70.93, 21.58, 20.98. HRMS (M+NH ⁴⁺ ) calc'd: 458.1268; observed: 458.1279.

Example 2

3-((((4-fluorophenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate

-357)

[00206] (5.76 mg, >97% pure). 'H NMR (CDCI ₃ ): δ 2.32 (s, 3 H, CH ₃ ); 5.15 (s, 2 H, OCH ₂ ); 7.11 (m, 1 H, Ar), 7.19 (m, 5 H, Ar); 7.41 (m, 2 H, Ar); 7.68 (m, 1 H, Ar); 7.94 (dd, 2H, J = 6.8, 4.8 Hz, Ar); 8.22 (dd, 1 H, J = 8.0, 2.0 Hz, Ar). C NMR (CDC1 ₃ ) δ: 169.70, 163.81 (d, ^ = 297 Hz), 162.50, 151.27, 150.98, 145.02, 138.86, 134.86, 134.83, 132.18, 130.78 (d, ³ JCF = 12.8 Hz), 129.94, 126.25, 126.03, 124.10, 122.57, 121.80, 1 16.75 (d, ² J _CF = 30.2 Hz), 70.30, 21.01. HRMS (M+NH ⁴⁺ ) calc'd: 462.1017; observed: 462.1024.

Example 3

3-((((4-acetylphenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate

(GTSm-304)

[00207] (7.39 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.30 (s, 3 H, CH ₃ ); 2.64 (s, 3 H, CH ₃ ); 5.16 (s, 2 H, OCH ₂ ); 7.06 (t, 1 H, J = 1.6 Hz, Ar), 7.18 (m, 3 H, Ar); 7.39 (m, 2 H, Ar); 7.66 (m, 1 H, Ar); 7.99 (AB dd, 2 H, J = 8.4, 1.6 Hz, Ar); 8.07 (AB dd, 2 H, J = 8.6, 1.6 Hz, Ar); 8.18 (dd, 1 H, J = 8.0, 1.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 195.55, 169.68, 162.71, 151.24, 150.69, 140.88, 140.08, 134.85, 134.66, 132.17, 130.00, 129.00, 128.28, 126.27, 126.12, 124.09, 122.65, 122.22, 121.85, 71.69, 28.89, 21.02; HRMS (M+NH ⁴⁺ ) calc'd: 486.1217; observed: 486.1224.

Example 4

3 -((2-acetoxybenzoyl)oxy)benzyl 2-acetoxybenzoate

[00208] (6.17 mg, >97% pure). 'H NMR (CDC1 ₃ ): δ 2.20 (s, 3 H, CH ₃ ); 2.31 (s, 3 H, CH ₃ ); 5.33 (s, 2 H, OCH ₂ ); 7.10 (d, J = 7.8 Hz, 1 H, Ar); 7.18 (d, 2 H, J = 8.1 Hz, Ar); 7.30-7.48 (m, 5 H, Ar); 7.55-7.68 (m, 2 H, Ar); 8.08 (dd, 1 H, J = 6.6, 1.2 Hz, Ar); 8.23 (dd, 1 H, J = 6.6, 1.2 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.72, 164.28, 162.91, 151.29, 150.82, 137.56, 134.75, 134.12, 132.25, 132.01, 129.92, 126.27, 126.13, 126.00, 124.11, 123.95, 123.12, 122.50, 121.84, 121.60, 66.28, 21.05, 20.87. HRMS (M+NH ⁴⁺ ) calc'd: 466.1496; observed: 462.1498. Example 5

3-((((4-chlorophenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate

-414)

[00209] (11.29 mg, >97% pure). ¾ NMR (CDC1 ₃ ): δ 2.31 (s, 3 H, CH ₃ ); 5.14 (s, 2 H, OCH ₂ ); 7.10 (m, 1 H, Ar); 7.16 (m, 3 H, Ar); 7.41 (m, 2 H, Ar); 7.50 (m, 2 H, Ar); 7.67 (dt, 1 H, J = 7.8, 1.6 Hz, Ar); 7.84 (dd, 2 H, J = 6.8, 2.0 Hz, Ar); 8.20 (d, 1 H, J = 1.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.66, 162.69, 151.23, 150.67, 140.53, 134.79, 134.74, 134.71, 132.16, 129.95, 129.51, 129.33, 126.22, 126.03, 124.06, 122.57, 122.20, 121.08, 71.41, 20.99. HRMS (M+NH ⁴⁺ ) calc'd: 478.0722; observed: 478.0730.

Example 6

4-((((4-fluorophenyl)sulfonyl)oxy)metriyl)phenyl 2-acetoxybenzoate

-357)

[00210] (5.46 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.30 (s, 3 H, CH ₃ ); 5.12 (s, 2 H, OCH ₂ ); 7.17 (m, 5 H, Ar); 7.32 (d, 2 H, J = 8.8 Hz Ar); 7.39 (dt, 1 H, J = 7.7, 1.2 Hz, Ar); 7.65 (dt, 1 H, J = 8.0, 1.7 Hz, Ar); 7.90 (m, 2 H, Ar); 8.20 (dd, 1 H, J = 7.9, 1.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.65, 165.41 (d, ¹ J _CF = 290 Hz), 162.66, 151.22, 151.14, 134.78, 132.45, 132.17, 130.71 (d, ³ J _CF = 12.6 Hz), 130.63, 130.03, 126.21, 124.05, 122.23, 122.14, 1 16.64 (d, ² JCF = 30.2 Hz), 71.50, 20.96. HRMS (M+NH ⁴⁺ ) calc'd: 462.1017; observed: 462.1003.

Example 7

4-((2-acetoxybenzoyl)oxy)benzyl 2-acetoxybenzoate

(ASApASA)

[00211] (6.29 mg, >97% pure). ^X H NMR (CDC1 ₃ ): 5 2.19 (s, 3 H, CH ₃ ); 2.31 (s, 3 H, CH ₃ ); 5.32 (s, 2 H, OCH ₂ ); 7.10 (dd, J = 8.0, 1.2 Hz, 1 H, Ar); 7.19 (m, 3 H, Ar); 7.32 (t, 1 H, J = 6.4 Hz, Ar); 7.40 (t, 1 H, J = 7.6 Hz, Ar); 7.50 (d, 2 H, J = 8.0 Hz, Ar); 7.57 (t, 1 H, J = 7.8 Hz, Ar); 7.65 (t, 1 H, J = 7.8 Hz, Ar); 8.07 (dd, 1 H, J = 8.0, 1.6 Hz, Ar); 8.23 (d, 1 H, J = 8.0, 1.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.83, 164.41, 162.98, 151.32, 150.84, 150.70, 134.83, 134.18, 133.63, 132.33, 132.08, 129.96, 126.34, 126.20, 124.17, 124.01, 123.20, 122.51, 122.13, 66.43, 21.13, 20.94. HRMS (M+NH ⁴⁺ ) calc'd: 466.1496; observed: 462.1491.

Example 8

4-((2-acetoxybenzoyl)oxy)benzyl ethyl maleate

-ME)

[00212] (8.55 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 1.28 (t, 3 H, J = 7.0 Hz, CH ₃ ); 2.31 (s, 3 H, CH ₃ ); 4.20 (q, 2 H, J = 14.1, 7.0 Hz, COOCH ₂ ); 5.24 (s, 2 H, OCH ₂ ); 6.28 (s, 2 H, HC=CH); 7.19 (m, 3 H, Ar); 7.41 (m, 2 H, Ar); 7.44 (m, 2 H, Ar); 7.66 (t, 1 H, J = 7.6 Hz, Ar); 8.22 (d, 1 H, J = 8.0 Hz, Ar). ¹³ C NMR (CDCI ₃ ) δ: 171.52, 165.20, 165.01, 162.90, 151.27, 150.67, 134.75, 133.24, 132.25, 130.61, 129.97, 129.20, 126.26, 124.11, 122.49, 121.98, 66.41, 61.36, 21.04, 14.04. HRMS (M+NH ⁴⁺ ) calc'd: 430.1496; observed: 430.1484.

Example 9

4-((2-acetoxybenzoyl)oxy)benzyl ethyl fumarate

(GTCp-FE)

[00213] (5.83 mg, >97% pure). 'H NMR (CDCI3): δ 1.29 (t, 3 H, J = 7.0 Hz, CH ₃ ); 2.31 (s, 3 H, CH ₃ ); 4.26 (q, 2 H, J = 14.2, 7.0 Hz, COOCH ₂ ); 5.25 (s, 2 H, OCH ₂ ); 6.89 (s, 2 H, HC=CH); 7.19 (m, 3 H, Ar); 7.42 (m, 3 H, Ar); 7.65 (m, 1 H, Ar); 8.22 (dd, 1 H, J = 7.8, 1.2 Hz, Ar). ¹³ C NMR (CDCI ₃ ) δ: 169.70, 164.86, 164.75, 162.89, 151.28, 150.69, 134.76, 134.32, 133.26, 133.16, 132.25, 129.81, 126.26, 124.1 1, 122.47, 122.04, 66.40, 61.43, 21.05, 14.15. HRMS (M+NH ⁴⁺ ) calc'd: 430.1496; observed: 430.1477.

Example 10

4-(2-bromoethyl)phenyl 2-acetoxybenzoate

-pBr)

[00214] (5.64 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.31 (s, 3 H, CH ₃ ); 3.20 (t, 2 H, J = 7.6 Hz, ArCH2); 3.57 (t, 2 H, J = 7.6 Hz, CH2Br); 7.12 (d, 2 H, J = 8.5 Hz, Ar); 7.16 (dd, 1 H, J = 8.1, 1.0 Hz, Ar); 7.26 (d, 2 H, J = 8.5 Hz, Ar); 7.38 (dt, 1 H, J = 7.7, 1.1 Hz, Ar); 7.63 (dt, 1 H, J = 8.0, 1.6 Hz, Ar); 8.22 (dd, 1 H, J = 7.9, 1.7 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ:

169.70, 162.96, 151.19, 149.43, 136.74, 134.62, 132.21, 129.82, 126.19, 124.03, 122.51, 121.82, 38.78, 32.65, 21.05. HRMS (M+NH ⁴⁺ ) calc'd: 380.0492; observed: 380.0473.

Example 1 1

4-(2-(tosyloxy)ethyl)phenyl 2-acetoxybenzoate

-pTs)

[00215] (8.95 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.31 (s, 3 H, CH ₃ ); 2.44 (s, 3 H, CH ₃ ); 2.98 (t, 2 H, J = 7.2 Hz, CH2); 4.21 (t, 2 H, J = 6.9 Hz, OCH ₂ ); 7.07 (d, 2 H, J = 8.4 Hz, Ar); 7.17 (m, 2 H, Ar); 7.31 (d, 2 H, J = 8.1 Hz, Ar); 7.40 (dt, 1 H, J = 7.6, 0.8 Hz, Ar); 7.65 (m, 1 H, Ar); 7.69 (d, 2H, J = 8.1 Hz, Ar); 8.21 (dd, 1 H, J = 7.6, 1.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.66, 162.95, 151.20, 149.49, 144.82, 134.68, 134.22, 133.93, 132.85, 132.16, 130.07, 129.89, 127.83, 126.23, 124.06, 122.53, 70.40, 34.72, 21.61, 21.03. HRMS (M+NH ⁴⁺ ) calc'd: 472.1425; observed: 472.1407.

Example 12

4-(2-((methylsulfonyl)oxy)ethyl)phenyl 2-acetoxybenzoate -pMS)

[00216] (8.65 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.31 (s, 3 H, CH ₃ ); 2.90 (s, 3 H, CH ₃ ); 3.09 (t, 2 H, J = 6.8 Hz, CH2); 4.43 (t, 2 H, J = 6.8 Hz, OCH ₂ ); 7.14 (m, 3 H, Ar); 7.29 (d, 2 H, J = 8.4 Hz, Ar); 7.40 (dd, 1 H, J = 7.6, 0.8 Hz, Ar); 7.65 (dt, 1 H, J = 7.6, 1.6 Hz, 1 H, Ar); 8.22 (dd, 1 H, J = 8.0, 1.6 Hz Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.78, 163.06, 151.25, 149.67, 134.76, 134.36, 132.26, 130.25, 126.30, 124.12, 122.54, 122.08, 70.10, 37.45, 35.13, 21.10. HRMS (M+NH ⁴⁺ ) calc'd: 396.1 112; observed: 396.11 17.

Example 13

3-((((4-isopropylphenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate

[00217] (7.85 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 1.27 (d, 6 H, J = 6.9 Hz, (CH ₃ )2CH) 2.30 (s, 3 H, CH ₃ ); 2.98 (sep, 1 H, J = 6.9 Hz, (CH ₃ )2CH), 5.09 (s, 2 H, OCH ₂ ); 7.38 (AB dd, 4 H, J = 17.6, 9.6 Hz, Ar); 7.65 (m, 1 H, Ar); 7.84 (d, 2 H, J = 8.3 Hz, Ar); 8.20 (dd, 1 H, J = 7.8, 1.4 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.69, 162.74, 155.60, 151.25, 150.64, 135.23, 134.77, 133.29, 132.18, 129.87, 128.15, 127.41, 126.22, 125.93, 124.09, 122.35, 122.29, 121.62, 70.88, 34.29, 23.58, 21.02. HRMS calc'd: 486.1581; observed: 486.1604.

Example 14

4-((((4-acetylphenyl)sulfonyl)oxy)methyl)phenyl 2-acetoxybenzoate

-304)

[00218] (5.86 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.30 (s, 3 H, CH ₃ ); 2.66 (s, 3 H, CH ₃ ); 5.15 (s, 2 H, OCH ₂ ); 7.12 (d, 2 H, J = 8.6 Hz, Ar); 7.18 (dd, 1H, J = 8.1 , 1.0 Hz, Ar); 7.32 (d, 2 H, J = 8.6 Hz, Ar); 7.39 (dt, 1H, J = 7.6, 1.0 Hz, Ar); 7.65 (dt, 2H, J = 7.8, 1.6 Hz, Ar); 7.98 (d, 2 H, J = 8.7 Hz, Ar); 8.08 (d, 2 H, J = 8.7 Hz, Ar); 8.19 (dd, 1 H, J = 7.8, 1.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 196.55, 169.64, 162.66, 151.19, 140.83, 140.19, 134.80, 132.16, 130.70, 130.11, 128.95, 128.17, 126.22, 124.05, 122.21, 122.14, 71.90, 26.88, 20.98. HRMS

(M+NH ⁴⁺ ) calc'd: 486.1217; observed: 486.1208.

Example 15

4-(((2-hydroxyethyl)thio)methyl)phenyl 2-acetoxybenzoate

-BME)

[00219] To a vigorously stirred solution of 4-(bromomethyl)phenyl 2-acetoxybenzoate (51 mg, 0.146 mmol) in anhydrous DMF (1.5 mL) under argon atmosphere sodium bicarbonate (25 mg, 0.292 mmol) was added at room temperature, followed by addition of 2- mercaptoethanol (10 \L, 1 1.4 mg, 0.146 mmol). The reaction mixture was stirred for 16 hours at to room temperature, then immersed in ice-water bath, then diluted with ethyl acetate (10 mL) and washed with water (5 mL). Organic layer was separated, dried over Na ₂ S0 ₄ , and concentrated under reduced pressure. The residue was purified by column

chromatography (S1O ₂ , hexane-ethyl acetate 10: 1) to afford 41 mg (81%) of product.

[00220] (7.85 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 1.33 (t, 1 H, J = 7.2 Hz, OH), 2.32 (s, 3 H, CH ₃ ); 2.66 (t, 2 H, J = 5.9 Hz, CH2); 3.71 (t, 2 H, J = 5.9 Hz, CH2); 3.75 (s, 2 H, ArCH2S); 7.14 (d, 2 H, J = 8.2 Hz, Ar); 7.17 (m, 1H, Ar); 7.40 (m, 3 H, Ar); 7.65 (t, 1 H, J = 7.6 Hz, Ar); 8.22 (d, 1 H, J = 7.7 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.72, 162.92, 151.17, 149.57, 135.94, 134.63, 132.19, 130.70, 130.00, 126.19, 124.02, 122.47, 121.80, 60.30, 35.13, 34.35, 21.01. HRMS (M+Na+) calc'd: 369.0773; observed: 369.0765.

Example 16

2-(((2-(nitrooxy)ethyl)thio)carbonyl)phenyl acetate

(GT-1031) [00221] To a solution of VLl-117 (53 mg, 0.175 mmol) in anhydrous acetonitrile (5 mL) silver nitrate (59 mg, 0.350 mmol) was added. The reaction mixture was stirred at reflux in dark under argon for 3 hours, then at +45 °C for 18 hours. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography (S1O ₂ , hexane- ethyl acetate 10: 1) to afford 17 mg (34%) of product.

[00222] (5.10 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.35 (s, 3 H, CH ₃ ); 3.34 (t, 2 H, J = 6.5 Hz, CH2), 4.63 (t, 2 H, J = 6.5 Hz, CH2); 7.29 (dd, 1 H, J = 7.9, 1.0 Hz, Ar); 7.35 (dt, 1 H, J = 7.7, 1.0 Hz, Ar); 7.59 (dt, 1 H, J = 7.7, 1.6 Hz, Ar); 7.31 (dd, 1 H, J = 7.9, 1.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 188.66, 169.33, 148.09, 134.16, 129.67, 129.41, 126.25, 124.06, 70.77, 26.19, 21.09. HRMS (M+Na+) calc'd: 308.0200; observed: 308.0198.

Example 17

l-(4-(bromomethyl)phenoxy)-2,4-dinitrobenzene

-DNP-001)

[00223] To a solution of VAL- 1-120 (205 mg, 0.706 mmol) in anhydrous chloroform (8 mL) chilled at 0°C polymer-supported triphenylphosphine (351 mg, 3 mmol per gram, 0.989 mmol) was added. The suspension was stirred for 10 minutes followed by addition of carbon tetrabromide (351 mg, 1.059 mmol) in anhydrous chloroform (2 mL). The reaction mixture was stirred for 16 hours while gradually warming up to room temperature. The mixture was filtered, concentrated under reduced pressure, and the residue was purified by flash column chromatography on silica gel (hexane-ethyl acetate 10: 1) to afford 158 mg (63%) of product.

[00224] (5.49 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 4.53 (s, 2 H, CH2); 7.09 (d, 1 H, J = 9.2 Hz, Ar); 7.12 (d, 2 H, J = 8.5 Hz, Ar); 7.52 (d, 2 H, J = 8.5 Hz, Ar); 8.35 (dd, 1 H, J = 9.2, 2.7 Hz, Ar), 8.86 (d, 1 H, J = 2.7 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 155.67, 153.51, 141.67, 139.70, 136.25, 131.41, 128.80, 122.1 1, 120.70, 118.78, 32.05. HRMS molecular ion is not observed.

Example 18

4-(2,4-dinitrophenoxy)benzyl nitrate

(GT-DNP-002)

[00225] To a solution of VL1-123 (108 mg, 0.306 mmol) in anhydrous acetonitrile (9 mL) silver nitrate (104 mg, 0.612 mmol) was added. The reaction mixture was stirred at reflux in dark under argon for 3 hours, then at +60 °C for 18 hours. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography (S1O ₂ , hexane- ethyl acetate 8: 1) to afford 101 mg (98%) of product.

[00226] (7.95 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 5.47 (s, 2 H, OCH ₂ ), 7.09 (d, 1 H, J = 9.2 Hz, Ar); 7.20 (d, 2 H, J = 8.6 Hz, Ar); 7.54 (d, 2 H, J = 8.6 Hz, Ar); 8.36 (dd, 1 H, J = 9.2, 2.7 Hz, Ar), 8.85 (d, 1 H, J = 2.7 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 155.44, 153.57, 141.83, 139.79, 131.61, 130.59, 128.89, 122.12, 120.72, 119.03, 73.66. HRMS molecular ion is not observed.

Example 19

4-(2,4-dinitrophenoxy)benzyl 2-acetoxybenzoate

(DNP-pASA)

[00227] To a vigorously stirred solution of VL1-120 (100 mg, 0.345 mmol) in anhydrous THF (2 mL) under argon atmosphere DMAP (4 mg, 0.035 mmol) was added. The mixture was chilled at 0 °C, and triethylamine (145 μί,, 104 mg, 1.035 mmol) was added. The reaction mixture was stirred for 5 minutes followed by dropwise addition of a solution of acetylsalicyloyl chloride (75 mg, 0.379 mmol) in anhydrous THF (1 mL). The reaction mixture was stirred for 16 hours while gradually warming up to room temperature. The solvent was removed under reduced pressure, and the mixture was diluted with water (10 mL), and extracted with dichloromethane (3 x 20 mL). Combined organic extracts were washed with brine, dried over Na ₂ S0 ₄ , evaporated, and the residue was purified by column chromatography (S1O ₂ , hexane-ethyl acetate 4: 1) to afford 1 15 mg (74%) of product.

[00228] (7.12 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.24 (s, 3 H, CH ₃ ); 5.33 (s, 2 H, CH2), 7.06 (d, 1 H, J = 9.3 Hz, Ar); 7.11 (dd, 1 H, J = 8.1, 0.8 Hz, Ar); 7.16 (d, 2 H, J = 8.6 Hz, Ar); 7.32 (dt, 1 H, J = 7.9, 0.8 Hz, Ar); 7.54 (d, 2 H, J = 8.6 Hz, Ar); 7.56 (m, 1 H, J = 8.1, 1.6 Hz, Ar); 8.05 (dd, 1 H, J = 7.9, 1.6 Hz, Ar); 8.31 (dd, 1 H, J = 9.3, 2.7 Hz, Ar), 8.82 (d, 1 H, J = 2.7 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.60, 164.13, 155.81, 153.60, 150.75, 141.58, 139.61, 134.19, 134.17, 131.78, 130.77, 128.82, 126.09, 123.91, 122.92, 122.07, 120.61, 118.72, 65.85, 20.90. HRMS (M+Na+) calc'd: 475.0748; observed: 475.0766.

Example 20

4-(((2-acetoxybenzoyl)oxy)methyl)naphthalen- 1 -yl 2-acetoxybenzoate

(MV77)

[00229] Under a positive pressure of argon and free of moisture, a solution of VAL-1-131 (20 mg, 0.060 mmol), 4-dimethylaminopyridine (2 mg, 0.016 mmol) and triethylamine (10 mg, 0.010 mmol) in anhydrous tetrahydrofuran (2 mL) was cooled to 0 °C, and a solution of ASA-C1 (14 mg, 0.179 mmol) in anhydrous tetrahydrofuran (2 mL) was added dropwise over a period of 10 min. The resulting solution was stirred at 0-5 °C for 4 hours. The reaction mixture was diluted with ethyl acetate (50 mL) and the diluted solution was washed with brine (20 mL x 3), dried over sodium sulfate, evaporated to dryness, and the residue was purified by column chromatography on silica gel (hexane/ethyl acetate = 4: 1) to give 7.9 mg (26%) of compound as a white solid.

[00230] (7.85 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.01 (s, 3 H, CH ₃ ); 2.62 (s, 3 H, CH ₃ ); 5.76 (s, 2 H, OCH ₂ ); 7.08 (dd, 1 H, J = 8.0, 1.0 Hz, Ar); 7.24 (dd, 1 H, J = 8.0, 1.2 Hz, Ar), 7.29 (dd, 1 H, J = 8.4, 1.2 Hz, Ar), 7.34 (d, 1 H, J = 7.6 Hz, Ar), 7.47 (dt, 1 H, J = 7.6, 1.2 Hz, Ar); 7.57 (m, 3 H, Ar), 7.65 (d, 1 H, J = 8.0 Hz, Ar), 7.71 (m, 1 H, Ar), 8.01 (m, 1 H, Ar), 8.03 (dd, 1 H, J = 8.0, 1.6 Hz, Ar), 8.08 (d, 1 H, J = 8.0 Hz, Ar), 8.40 (dd, 1 H, J = 8.0, 2.0 Hz, Ar). ¹³ C NMR (CDCI ₃ ) δ: 169.87, 169.76, 164.53, 163.00, 151.57, 150.90, 147.58, 135.04, 134.17, 133.12, 132.48, 132.18, 129.77, 127.77, 127.60, 127.30, 127.00, 126.49, 126.18, 124.40, 124.18, 124.03, 123.20, 122.43, 122.16, 117.84, 65.09, 21.12, 20.71. HRMS (M+Na+) calc'd: 521.1212; observed: 521.1215.

Example 21

2-(((4-(bromomethyl)phenyl)thio)carbonyl)phenyl acetate

(GTth-pBr)

[00231] VL1-133 (0.96 g, 3.19 mmol) was dissolved in anhydrous chloroform (5 mL), followed by addition of polymer-supported triphenylphosphine (1.49 g, 3 mmol per gram, 4.45 mmol). The suspension was chilled at 0°C followed by addition of carbon tetrabromide (1.59 g, 4.79 mmol). The reaction mixture was stirred for 16 hours while gradually warming up to room temperature. The mixture was filtered, concentrated under reduced pressure, and the residue was purified by flash column chromatography on silica gel (ethyl acetate eluting in hexane from 10% to 33%) to afford 0.78 g (67%) of product as a white solid.

[00232] (9.23 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.32 (s, 3 H, CH ₃ ); 4.51 (s, 2 H, BrCH2); 7.16 (dd, 2 H, J = 7.9, 1.0 Hz, Ar); 7.37 (dt, 2 H, J = 7.6, 1.0 Hz, Ar); 7.47 (s, 4 H, Ar); 7.59 (dt, 1 H, J = 7.6, 1.6 Hz, Ar); 8.22 (dd, 1 H, J = 7.9, 1.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 187.76, 169.31, 148.16, 135.25, 135.24, 133.96, 129.88, 129.76, 129.65, 127.67, 126.23, 124.13, 32.51, 21.14. HRMS (M+Na+) calc'd: 369.0773; observed: 369.0765.

Example 22

2-(((4-((nitrooxy)methyl)phenyl)thio)carbonyl)phenyl acetate

-pNO)

[00233] To a solution of VL1-134 (206 mg, 0.564 mmol) in anhydrous acetonitrile (17 mL) silver nitrate (192 mg, 1.128 mmol) was added. The mixture was stirred at reflux in dark under argon for 3 hours. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography (S1O ₂ , hexane-ethyl acetate 8: 1) to afford 126 mg (64%>) of product.

[00234] (6.92 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.33 (s, 3 H, CH ₃ ); 5.47 (s, 2 H, OCH ₂ ); 7.16 (dd, 2 H, J = 8.1, 1.0 Hz, Ar); 7.38 (dt, 2 H, J = 7.7, 1.0 Hz, Ar); 7.47 (d, 2 H, J = 8.2 Hz, Ar); 7.54 (d, 2 H, J = 8.2 Hz, Ar); 7.60 (dt, 1 H, J = 7.8, 1.5 Hz, Ar); 8.04 (dd, 1 H, J = 7.8, 1.5 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 187.55, 169.31, 148.18, 135.34, 134.05, 133.80, 129.76, 129.58, 129.02, 126.25, 124.13, 73.89, 21.13. HRMS (M+Na+) calc'd: 370.0356; observed: 370.0362. Example 23

4-((2-acetoxybenzoyl)thio)benzyl 2-acetoxybenzoate

(GTth-pASA)

[00235] To a vigorously stirred solution of VL1-133 (100 mg, 0.249 mmol) in anhydrous THF (3 mL) under argon atmosphere DMAP (6 mg, 0.050 mmol) was added. The mixture was immersed stirred into a dry ice-acetone bath (-78 °C), and DIPEA (162 μΕ, 121 mg, 0.933 mmol) was added. The reaction mixture was stirred for 5 minutes followed by dropwise addition of a solution of acetylsalicyloyl chloride (76 mg, 0.383 mmol) in anhydrous THF (2 mL). The reaction mixture was stirred at -78 °C for 1 hour, then at 0 °C for 3 hours. The cold bath was removed, and the mixture was stirred for 16 hours while gradually warming up to room temperature. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (S1O ₂ , hexane-ethyl acetate 4: 1) to afford 88 mg (57%) of product GTth-pASA.

[00236] (8.43 mg, >97% pure). ^X H NMR (CDC1 ₃ ): 5 2.19 (s, 3 H, CH ₃ ); 2.31 (s, 3 H, CH ₃ ); 5.35 (s, 2 H, OCH ₂ ); 7.11 (d, 1 H, J = 8.0 Hz, Ar); 7.17 (d, 1 H, J = 8.0 Hz, Ar); 7.33 (t, 1 H, J = 8.7 Hz, Ar); 7.37 (t, 1 H, J = 7.6 Hz, Ar); 7.50 (AB d, 2 H, J = 8.5 Hz, Ar); 7.53 (AB d, 2 H, J = 8.5 Hz, Ar); 7.58 (m, 2 H, Ar); 8.04 (dd, 1 H, J = 7.8, 1.2 Hz, Ar), 8.06 (dd, 1 H, J = 7.8, 1.4 Hz, Ar). ¹³ C NMR (CDCI3) δ: 187.86, 169.72, 169.32, 164.25, 150.75, 148.15, 137.24, 135.26, 134.13, 133.96, 131.96, 129.76, 129.67, 129.07, 127.61, 126.25, 126.10, 124.13, 123.92, 123.02, 66.30, 21.13, 20.85. HRMS (M+NH ⁴⁺ ) calc'd: 482.1268; observed: 482.1283.

Example 24

2-(((4-(((2-hydroxyethyl)thio)methyl)phenyl)thio)carbonyl)ph enyl acetate

-pBME)

[00237] To a vigorously stirred solution of VL1-134 (200 mg, 0.548 mmol) in anhydrous DMF (3 mL) under argon atmosphere sodium bicarbonate (97 mg, 1.150 mmol) was added at room temperature, followed by addition of 2-mercaptoethanol (40 \L, 45 mg, 0.585 mmol). The reaction mixture was stirred for 20 hours at to room temperature, then immersed in ice- water bath, then diluted with ethyl acetate (20 mL) and washed with water (10 mL). Organic layer was separated, dried over Na ₂ S0 ₄ , and concentrated under reduced pressure. The residue was purified by column chromatography (S1O ₂ , hexane-ethyl acetate 3 : 1) to afford 28 mg (14%) of product.

[00238] (5.25 mg, >97% pure). ^X H NMR (CDC1 ₃ ): 2.31 (s, 3 H, CH ₃ ); δ 2.61 (t, 1 H, J = 5.2 Hz, OH), 2.65 (t, 2 H, J = 5.9 Hz, CH2); 3.70 (t, 2 H, J = 5.9 Hz, CH2); 3.76 (s, 2 H, ArCH2S); 7.16 (d, 2 H, J = 8.0 Hz, Ar); 7.40 (m, 5 H, Ar); 7.59 (t, 1 H, J = 7.7 Hz, Ar); 8.03 (d, 1 H, J = 7.7 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 188.14, 162.34, 148.13, 139.90, 135.14, 133.87, 130.42, 129.78, 129.42, 126.21, 126.13, 124.09, 60.31, 35.44, 34.40, 21.15. HRMS molecular ion is not observed.

Example 25

4-(bromomethyl)naphthalen- 1 -yl 2-acetoxybenzoate

-1-96)

[00239] To a solution of VAL-1-131 (120 mg, 0.357 mmol) in anhydrous chloroform (3 mL) chilled to 0 °C (ice water bath) under argon atmosphere, polymer supported

triphenylphosphine (434 mg, 2.5 mmol) was added. The mixture was stirred for 15 minutes, followed by addition of solution of carbon tetrabromide (445 mg, 2.5 mmol) in chloroform (1 mL). The reaction mixture was stirred for 48 h while gradually warming up to room temperature. Reaction mixture was concentrated and purified by column chromatography on silica gel (gradually eluting ethyl acetate in hexane from 5 to 15%) to afford 88 mg (62%) of product.

[00240] (6.92 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.27 (s, 3 H, CH ₃ ); 4.98 (s, 2 H, CH2Br); 7.24 (m, 2 H, Ar); 7.48 (m, 1 H, Ar); 7.58 (m, 2 H, Ar); 7.70 (m, 2 H, Ar); 8.01 (d, 1 H, J = 8.0 Hz, Ar); 8.21 (d, 1 H, J = 8.4 Hz, Ar); 8.39 (d, 1 H, J = 7.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) 5: 169.39, 162.40, 151.05, 147.04, 134.56, 131.98, 131.89, 131.29, 127.14, 127.06, 126.87, 126.60, 125.99, 123.89, 123.82, 121.85, 121.76, 117.45, 30.76, 20.63. HRMS (M+NH ⁴⁺ ) calc'd: 416.0492; observed: 416.0500. Example 26

4-((nitrooxy)methyl)naphthalen- 1 -yl 2-acetoxybenzoate

(RG-1-99)

[00241] To a solution of RG- 1-96 (67 mg, 0.17 mmol) in anhydrous acetonitrile (7 mL), silver nitrate (57 mg, 0.34 mmol) was added. The reaction mixture (covered with Aluminium foil) was stirred at reflux overnight under argon. Reaction was clean and there was no need to purify it. Obtained 46 mg (71%) of product.

[00242] (6.89 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.28 (s, 3 H, CH ₃ ); 5.92 (s, 2 H, OCH ₂ ); 7.26 (m, 1 H, Ar); 7.37 (d, 1 H, J = 7.7 Hz, Ar); 7.47 (m, 1 H, Ar); 7.60 (m, 2 H, Ar); 7.66 (d, 1 H, J = 7.7 Hz, Ar); 7.67 (m, 1 H, Ar); 7.73 (m, 2 H, Ar); 8.04(t, 2 H, J = 8.5 Hz, Ar); 8.40 (dd, 1 H, J = 7.8, 0.5 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.38, 162.36, 151.08, 147.86, 134.65, 132.58, 131.97, 128.71, 127.48, 126.88, 126.70, 126.01, 125.69, 123.91, 123.25, 121.85, 121.75, 1 17.26, 72.30, 20.62. HRMS (M+NH ⁴⁺ ) calc'd: 404.0741 ; observed:

404.0758.

Example 27

4-((2-acetoxybenzoyl)thio)benzyl acetate

(VL-1-139F 1)

[00243] To a vigorously stirred solution of VL1-133 (100 mg, 0.249 mmol) in anhydrous THF (3 mL) under argon atmosphere DMAP (6 mg, 0.050 mmol) was added. The mixture was immersed stirred into a dry ice-acetone bath (-78 °C), and DIPEA (162 μΕ, 121 mg, 0.933 mmol) was added. The reaction mixture was stirred for 5 minutes followed by dropwise addition of a solution of acetylsalicyloyl chloride (76 mg, 0.383 mmol) in anhydrous THF (2 mL). The reaction mixture was stirred at -78 °C for 1 hour, then at 0 °C for 3 hours. The cold bath was removed, and the mixture was stirred for 16 hours while gradually warming up to room temperature. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (S1O ₂ , hexane-ethyl acetate 4: 1) to afford 25 mg (22%) of product GTth-pAc (VL-1-139F1).

[00244] (25 mg, >97% pure). ^X H NMR (CDC1 ₃ ): 5 2.13 (s, 3 H, CH ₃ ); δ 2.32 (s, 3 H, CH ₃ ); 5.15 (s, 2 H, OCH ₂ ); 7.16 (d, 2 H, J = 8.0 Hz, Ar); 7.38 (m, 1 H, Ar); 7.47 (m, 4 H, Ar); 7.59 (t, 1 H, J = 7.6 Hz, Ar); 8.22 (d, 1 H, J = 7.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 187.94, 170.75, 169.31, 148.16, 137.59, 135.10, 133.91, 129.76, 129.70, 128.89, 127.31, 126.22, 124.1 1, 65.58, 21.13, 20.96. HRMS (M+Na+) calc'd: 362.1057; observed: 362.1071.

Example 28

3 -(azidomethyl)-4-((nitrooxy)methyl)phenyl 2-acetoxybenzoate

-1-52)

[00245] To a solution of 3-azidomethyl-4-(2-acetoxybenzoyl)benzyl chloride (68 mg, 0.189 mmol) in anhydrous acetonitrile (3 mL) silver nitrate (71 mg, 0.416 mmol) was added. The mixture was stirred at +45 °C in dark under argon for 22 h. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography (Si0 ₂ , hexane-ethyl acetate 8: 1) to afford 53 mg (73%) of product.

[00246] (23 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.31 (s, 3 H, CH ₃ ); 4.47 (s, 2 H, CH2N3); 5.15 (s, 2 H, OCH ₂ ); 7.22 (m, 3 H, Ar); 7.40 (m, 1 H, Ar); 7.52 (d, 1 H, J = 8.3 Hz, Ar); 7.67 (m, 1 H, Ar); 8.22 (dd, 1 H, J = 7.9, 1.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.66, 162.53, 151.62, 151.29, 136.90, 134.94, 132.59, 132.17, 128.27, 126.25, 124.1 1, 123.18, 122.11, 122.06, 71.42, 51.84, 20.98. HRMS (M+Na+) calc'd: 409.0755; observed: 409.0753.

Example 29

3-(azidomethyl)-4-(bromomethyl)phenyl 2-acetoxybenzoate

-1-92)

[00247] To a vigorously stirred solution of triphenylphosphine (0.409 g, 1.56 mmol) in anhydrous DMF (2 mL) under argon atmosphere bromine (82 μΐ _^ , 1.60 mmol) was added at 0 °C. The reaction mixture was stirred for 30 min while gradually warming up to room temperature. The reaction mixture was then immersed in ice-water bath, and a solution of 3- azidomethyl-4-(2-acetoxybenzoyl)benzyl alcohol (0.162 g, 0.459 mmol) in anhydrous DMF (1 mL) was added. The mixture was stirred at +60 °C under argon for 16 h, then cooled down to room temperature and concentrated under reduced pressure. The residue was purified by column chromatography (S1O ₂ , hexane-ethyl acetate 10: 1) to afford 134 mg (72%) of product.

[00248] (18 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.31 (s, 3 H, CH ₃ ); 4.55 (s, 4 H, CH2N3, CH2Br); 7.18 (m, 3 H, Ar); 7.39 (m, 1 H, Ar); 7.45 (d, 1 H, J = 8.3 Hz, Ar); 7.65 (m, 1 H, Ar); 8.20 (dd, 1 H, J = 7.9, 1.4 Hz, Ar). ¹³ C NMR (CDCI3) δ: 169.67, 162.60, 151.26, 150.90, 136.14, 134.86, 133.65, 132.20, 132.19, 126.25, 124.10, 122.95, 122.19, 122.09, 51.42, 29.72, 21.01. HRMS (M+NH ⁴⁺ ) calc'd: 421.0506; observed: 421.0506.

Example 30

4-((2-acetoxybenzoyl)oxy)-2-(azidomethyl)benzyl 2-acetoxybenzoate

-2-24F2)

[00249] To a vigorously stirred solution of 3-azidomethyl-4-(2-acetoxybenzoyl)benzyl alcohol (85 mg, 0.249 mmol) in anhydrous THF (2 mL) under argon atmosphere DMAP (6 mg, 0.050 mmol) was added. The mixture was immersed stirred into a dry ice-acetone bath (- 78 °C), and DIPEA (130 μί,, 0.1 Al mmol) was added. The reaction mixture was stirred for 5 minutes followed by slow addition of a solution of acetylsalicyloyl chloride (59 mg, 0.299 mmol) in anhydrous THF (2 mL). The reaction mixture was stirred at -55 °C for 1 h, then the cold bath was removed, and the mixture was stirred for 18 h while gradually warming up to room temperature. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (S1O ₂ , hexane-ethyl acetate 10: 1) to afford 41 mg (33%) of 3-azidomethyl-4-(2-acetoxybenzoyl)benzyl (2-acetoxy)benzoate (VL2-24F2). [00250] (41 mg, >97% pure). 'H NMR (CDC1 ₃ ): δ 2.16 (s, 3 H, CH ₃ ); 2.31 (s, 3 H, CH ₃ ); 4.22 (s, 2 H, CH2N3), 5.37 (s, 2 H, OCH ₂ ); 7.10 (d, J = 7.9 Hz, 1 H, Ar); 7.20 (m, 2 H, Ar); 7.21 (m, 1 H, Ar); 7.32 (t, 1 H, J = 7.3 Hz, Ar); 7.40 (t, 1 H, J = 7.3 Hz, Ar); 7.57 (m, 2 H, Ar); 7.57 (t, 1 H, J = 7.8 Hz, Ar); 7.64 (m, 1 H, Ar); 8.04 (dd, 1 H, J = 7.8, 1.2 Hz, Ar); 8.23 (dd, 1 H, J = 7.8, 1.2 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.65, 164.10, 162.98, 162.68, 151.24, 150.97, 150.75, 136.47, 134.81, 134.19, 132.20, 131.89, 131.50, 126.23, 126.1 1, 124.07, 123.93, 122.86, 122.68, 122.23, 121.85, 63.82, 51.72, 20.98, 20.71. HRMS (M+NH ⁴⁺ ) calc'd: 521.1667; observed: 521.1641.

Example 31

4-(acetoxymethyl)-3 -(azidomethyl)phenyl 2-acetoxybenzoate

-2-24F1)

[00251] To a vigorously stirred solution of 3-azidomethyl-4-(2-acetoxybenzoyl)benzyl alcohol (85 mg, 0.249 mmol) in anhydrous THF (2 mL) under argon atmosphere DMAP (6 mg, 0.050 mmol) was added. The mixture was immersed stirred into a dry ice-acetone bath (- 78 °C), and DIPEA (130 μί, 0.747 mmol) was added. The reaction mixture was stirred for 5 minutes followed by slow addition of a solution of acetylsalicyloyl chloride (59 mg, 0.299 mmol) in anhydrous THF (2 mL). The reaction mixture was stirred at -55 °C for 1 h, then the cold bath was removed, and the mixture was stirred for 18 h while gradually warming up to room temperature. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (S1O ₂ , hexane-ethyl acetate 10: 1) to afford 37 mg (39%) of 3 -azidomethyl-4-(2-acetoxybenzoyl)benzyl (2-acetoxy)benzoate (VL2-24F 1 ).

[00252] (37 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 2.1 1 (s, 3 H, CH ₃ ); δ 2.31 (s, 3 H, CH ₃ ); 4.48 (s, 1 H, CH2N3), 5.17 (s, 2 H, OCH ₂ ); 7.20 (m, 3 H, Ar); 7.40 (m, 1 H, Ar); 7.49 (d, 1 H, J = 8.4 Hz, Ar); 7.66 (t, 1 H, J = 7.6 Hz, Ar); 8.22 (d, 1 H, J = 7.6 Hz, Ar). ¹³ C NMR (CDC1 ₃ ) 5: 170.57, 169.65, 162.71, 151.24, 150.76, 136.15, 134.81, 132.20, 131.96, 131.78, 126.22, 124.07, 122.71, 122.23, 121.79, 63.05, 51.74, 20.99, 20.92. HRMS [M+NH ₄ ] ⁺ for Ci ₉ H ₂₁ N ₄ 0 ₆ calc'd: 401.1456; observed: 401.1445.

Example 32 2-(ethoxycarbonyl)phenyl ethyl fumarate

(VL-2-43)

[00253] To a solution of ethyl salicylate (1.00 mL, 1.13 g, 6.80 mmol) in anhydrous dichloromethane (10 mL) chilled at 0 °C triethylamine (1.05 mL, 0.76 g, 7.48 mmol) was added. After stirring for 10 minutes, ethyl fumaroyl chloride (1 mL, 1.22 g, 7.48 mmol) was added dropwise. After stirring for 10 minutes at 0 °C the cold bath was removed, and the reaction mixture was stirred for 30 minutes while warming up to room temperature.

Saturated sodium bicarbonate solution (5 mL) was added. Organic layer was separated; the aqueous layer was extracted with dichloromethane (10 mL). Combined organic extracts were dried over Na ₂ S0 ₄ , filtered, mixed with silica gel (ca 5 g), evaporated, and the resulting solid was applied onto a chromatography column followed by purification (elution of ethyl acetate in hexane from 0 to 9%) to afford 1.55 g (78%) of product.

[00254] (17 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 1.29 (t, 3 H, J = 7.0 Hz, CH ₃ ); 1.36 (t, 3 H, J = 7.0 Hz, CH ₃ ); 4.20 (q, 3 H, J = 7.0 Hz, COOCH ₂ ); 4.26 (q, 2 H, J = 7.0 Hz, COOCH ₂ ); 6.31 (s, 2 H, HC=CH); 7.64 (m, 2 H, Ar); 7.81 (d, 1 H, J = 7.6, Hz, Ar); 8.02 (d, 1 H, J = 7.6, Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 166.73, 164.28, 164.15, 153.59, 134.88, 133.38, 131.40, 130.29, 125.41, 123.09, 1 19.94, 61.43, 60.90, 14.15, 14.11.

Example 33

bis(2-(ethoxycarbonyl)phenyl) fumarate

(VL-2-44)

[00255] To a solution of ethyl salicylate (1.00 mL, 1.13 g, 6.80 mmol) in anhydrous dichloromethane (10 mL) chilled at 0 °C triethylamine (1.05 mL, 0.76 g, 7.48 mmol) was added. After stirring for 10 minutes, fumaroyl chloride (0.33 mL, 0.47 g, 3.06 mmol) was added dropwise. After stirring for 5 minutes at 0 °C the cold bath was removed, and the reaction mixture was stirred for 5 hours while warming up to room temperature. Saturated sodium bicarbonate solution (5 mL) was added. Organic layer was separated; the aqueous layer was extracted with dichloromethane (10 mL). Combined organic extracts were dried over Na ₂ S0 ₄ , filtered, mixed with silica gel (ca 1 g), evaporated, and the resulting solid was applied onto a chromatography column followed by purification (elution of ethyl acetate in hexane from 9 to 1 1%) to afford 0.55 g (44%) or product.

[00256] (44 mg, >97% pure). ^X H NMR (CDC1 ₃ ): δ 1.29 (t, 6 H, J = 7.0 Hz, CH ₃ ); 4.30 (q, 4 H, J = 7.0 Hz, COOCH ₂ ); 6.31 (s, 2 H, HC=CH); 7.63 (m, 4 H, Ar); 7.80 (d, 2 H, J = 7.5, Hz, Ar); 8.02 (d, 2 H, J = 7.5, Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 164.27, 164.21, 153.55, 134.17, 133.38, 130.29, 125.44, 123.06, 1 19.86, 60.91, 14.1 1.

Example 34

bis(4-((2-acetoxybenzoyl)oxy)benzyl) fumarate

-2-45)

[00257] (15 mg, >97% pure). 'H NMR (CDC1 ₃ ): δ 2.29 (s, 6 H, CH ₃ ); 5.16 (s, 4 H, OCH ₂ ); 6.30 (s, 2 H, HC=CH); 7.17 (d, 4 H, J = 8.0, Hz, Ar); 7.30 (d, 4 H, J = 8.0, Hz, Ar); 7.67 (m, 4 H, Ar); 7.85 (d, 2 H, J = 7.8, Hz, Ar); 8.19 (d, 2 H, J = 7.8, Hz, Ar). ¹³ C NMR (CDC1 ₃ ) δ: 169.07, 169.03, 163.41, 150.75, 148.33, 134.26, 132.93, 132.37, 130.69, 128.70, 125.44, 124.36, 121.79, 120.88, 65.91, 20.31.

Example 35

-nitrophenyl)sulfonyl)ethyl 2-acetoxybenzoate

-pVS)

[00258] The compound of Example 35 is prepared according to methods disclosed herein. Example 36

4-((2-(ethoxycarbonyl)phenoxy)methyl)phenyl 2-acetoxybenzoate

[00259] Under a positive pressure of nitrogen and free of moisture, a solution of either 4-hydroxymethylphenol ester of 2-acetyloxybenzoic acid (500 mg, 1.75 mmol), 4- dimethylaminopyridine (21 mg, 0.18 mmol) and triethylamine (513 mg, 5.25 mmol) in tetrahydrofuran (10 mL) was cooled to 0 °C, and 2-chlorocarbonyl)phenyl acetate (2.00 mmol) was added dropwise over a period of 10 min. The resulting solution was stirred at 0-5 °C for 4 h. The reaction mixture was diluted with ethyl acetate (100 mL) and the diluted solution was washed with brine (50 mL x 3), dried over sodium sulfate, evaporated to dryness, and the residue was purified by column chromatography on silica gel.

[00260] Purified using AcOEt/hexane (20:80) as eluent to afford 250 mg (32%) of the desired product as an oil. ^X H NMR (CDC1 ₃ ): δ 8.23 (dd, 1 H, J = 8.0, 1.6 Hz, Ar), 8.07 (dd, 1 H, J = 8.0, 1.6 Hz, Ar), 7.65 (t, 1 H, J = 7.8 Hz, Ar), 7.57 (t, 1 H, J = 7.8 Hz, Ar), 7.50 (d, 2 H, J = 8.0 Hz, Ar), 7.40 (t, 1 H, J = 7.6 Hz, Ar), 7.32 (t, 1 H, J = 6.4 Hz, Ar), 7.19 (m, 3 H, Ar), 7.10 (dd, J = 8.0, 1.2 Hz, 1 H, Ar), 5.32 (s, 2 H, OCH ₂ ), 2.31 (s, 3 H, CH ₃ ), 2.19 (s, 3 H, CH ₃ ). ¹³ C NMR (CDCI ₃ ) δ: 169.83, 164.41, 162.98, 151.32, 150.84, 150.70, 134.83, 134.18, 133.63, 132.33, 132.08, 129.96, 126.34, 126.20, 124.17, 124.01, 123.20, 122.51, 122.13, 66.43, 21.13, 20.94. HRMS (M+NH ₄ ⁺ ) for C ₂ 5H ₂₄ N0 ₈ calc'd: 466.1496; observed:

466.1491.

Example 37

2-((2-(ethoxycarbonyl)phenoxy)methyl)phenyl 2-acetoxybenzoate

[00261] Under a positive pressure of nitrogen and free of moisture, a solution of either 2-hydroxymethylphenol ester of 2-acetyloxybenzoic acid (500 mg, 1.75 mmol), 4- dimethylaminopyridine (21 mg, 0.18 mmol) and triethylamine (513 mg, 5.25 mmol) in tetrahydrofuran (10 mL) was cooled to 0 °C, and 2-chlorocarbonyl)phenyl acetate (2.00 mmol) was added dropwise over a period of 10 min. The resulting solution was stirred at 0-5 °C for 4 h. The reaction mixture was diluted with ethyl acetate (100 mL) and the diluted solution was washed with brine (50 mL x 3), dried over sodium sulfate, evaporated to dryness, and the residue was purified by column chromatography on silica gel.

[00262] Purified using AcOEt/hexane (30:70) as eluent to afford 188 mg (24%) of the desired product as a white solid. ^X H NMR (CDC1 ₃ ): δ 8.22 (d, 1 H, J = 7.8 Hz, Ar), 8.00 (d, 1 H, J = 7.8 Hz, Ar), 7.42-7.65 (m, 4 H, Ar), 7.24-7.37 (m, 4 H, Ar), 7.17 (d, 1 H, J = 8.1 Hz, Ar), 7.08 (d, 1 H, J = 8.1 Hz, Ar), 5.33 (s, 2 H, OCH ₂ ), 2.85 (s, 3 H, CH ₃ ), 2.16 (s, 3 H, CH ₃ ). ¹³ C NMR (CDC1 ₃ ) δ: 169.73, 164.1 1, 162.66, 151.43, 150.84, 149.11, 134.87, 134.05, 132.22, 132.06, 130.53, 129.97, 128.12, 126.60, 126.33, 126.09, 124.14, 123.89, 123.00, 122.85, 122.13, 62.1 1, 21.04, 20.83. HRMS (M+NH ₄ ⁺ ) for C25H24 O ₈ calc'd: 466.1496; observed: 466.1498.

Example 38

3-((2-acetoxybenzoyl)oxy)benzyl ethyl fumarate

(GTCm-FE)

[00263] The compound of Example 38 was prepared according to methods disclosed herein.

Biological Activity

[00264] As shown in Table 1, compounds of the invention show in vitro biological activity, including: 1) anti-inflammatory activity: inhibition of LPS-induced upregulation of iNOS and cytokines in mouse RAW 264.7 macrophage cells; 2) phase 2 enzyme induction: upregulation of NQOl in Hepa lclc7 cells liver cells; 3) cytotoxicity in cancer cells, HepG2, Hepa lclc7, and macrophages; 4) activation of stress response in liver cells; and 5) bioactivation by esterase action.

Table 1

16 2.40 0.15 17.0

Example 353 yes 9.7 ± 1 10.2 ±3.2 1.87 ±0.75 85.0 ±2.0 17

Example 335 yes 2.4 ±0.4 39.6 ±1.5 6.40 ±0.18 23.0 ±3.0 18

Example 452 yes 4.2 ±0.6 48.6 ±0.2 5.46 ±0.50 3.0 ±0.1 19

Example 498 yes 22.2 ±0.4 38.7 ±4.3 1.74 ± 1.61 111.0 ± 20 13.0

Example 365 yes 66.9 ± 1 6.50 ± 1.8 1.52 ±0.04 63.0 ±0.0 21

Example 347 yes 44.1 ±0.4 0.40 ±0.2 1.32 ±0.01 91.0 ± 1.0 22

Example 464 yes 4.8 ±2.4 11.5 ± 1.4 3.25 ±0.77 31.0 ±3.0 23

Example 362 1.32 ±0.53 49.0 ± 5.0 24

Example 399 yes 17.6 ±3.8 37.4 ± 1.6 1.38 ±0.02 85.0 ±4.0 25

Example 381 yes 16.1 ±5 54.1 ±6.1 1.46 ±0.05 91.0 ± 1.0 26

Example 344 yes 1.4 ±0.2 4.8 ±0.7 1.33 ±0.09 87.0 ±5.0 27

Example 386 yes 69.2 ±0.9 8.50 ± 0.97 ±0.10 810 ±5.0 28 0.50

Example 404 yes 39.4 ±0.4 32.5 ±0.7 0.55 ±0.03 74.0 ± 2.0 29

[00265] The GSH conjugate assay was performed with lmM GSH, 0.375mg/mL PLE and 100 μΜ concentration of the drug. The GSH conjugate was identified by LC-MS as [M+H]+ [(m/z=414 for DNP-pASA, DNP-001& GT-DNP-002), (m/z= 430 for VL-1-139 Fl, GTth- pNO, GTth-pBr & GTthpASA), (m/z =464 for RG-1-96 and RG-1-99)]. Each compound was tested in duplicate. The ARE luciferase and SRB cytotoxicity assay was run in HepG2 cells, each compound tested at 20μΜ for 8 h. Data represents mean and s.e.m. of duplicates. The NQOl induction assay was run in Heplclc7 cells at 20 μΜ for 48 h. Data shows mean and s.d. for duplicates. The MTT assay was run in LPS-induced RAW 264.7 cells at 20 μΜ for 24 h with DMSO as the control. Data show mean and s.d. for duplicates.

Inhibition of the NF B Pathway [00266] Compounds of the invention show significant inhibition of NFKB activity on an artificial reporter construct and endogenous target genes, without toxicity towards breast cancer cells grown in a 2-dimensional monolayer but with significant inhibition of growth of cancer cells in a 3 -dimensional culture system. This 3D system enriches for cells with a cancer stem cell phenotype, suggesting that the compounds may be an effective therapy for targeting aggressive breast tumors.

[00267] The NFKB pathway is important in breast cancer biology by promoting cell survival, motility and invasion, tumor angiogenesis, cancer-stem cell (CSC) properties and therapeutic failure of both endocrine and chemotherapy - all phenotypes associated with a poor patient outcome. Therefore, inhibition of NFKB is desired in the clinical management of breast cancer.

[00268] GTCpFE (Example 9) was evaluated for inhibition of NFKB activity in breast cancer cells. GTCpFE showed inhibition of NFKB pathway activation in breast cancer cells by inhibiting nuclear translocation of the p65 and p50 transcription factors and their binding to DNA. GTCpFE can prevent formation of mammospheres, a surrogate read out for anti- breast CSC activity. This suggests that compounds of the invention may have anti-cancer properties by blocking self-renewal and anoikis resistance - both mechanisms required to promote aggressiveness in breast cancer.

[00269] GTCpFE inhibits NFKB activity in breast cancer cell lines. The effect of GTCpFE on proinflammatory cytokine-induced NFKB activity in breast cancer cells was examined using a transiently transfected NFKB-RE-driven reporter construct and QPCR for endogenous NFKB target genes. TNFcc-induced NFKB-RE activity in ER+ MCF-7 cells was reduced in a dose-dependent manner by GTCpFE with a calculated IC50 values of -20 μΜ (FIG. 1).

Expression of TNFcc-induced NFKB target genes (ICAM1, TNFcc, and MCP-1) was also inhibited by GTCpFE (FIG. 2). In addition, GTCpFE was found to prevent in other breast cancer cell lines, such as MDA-MB-231 (FIG. 3) and BT474 (FIG. 4), suggesting that GTCpFE is capable of inhibiting NFKB activity in breast cancer cells. GTCmFE (Example 38) inhibited TNF a- induced NFKB-RE reporter activity (FIG. 5) and expression of endogenous target genes (FIGS. 6A-6C) in a similar manner to GTCpFE.

[00270] GTCpFE inhibits the NFKB pathway, mammosphere formation and CD44+/CD24- population. Both cell survival and CSC properties are known to be affected by NFKB, therefore regulation of either by GTCpFE was evaluated. As a read-out for in vitro CSC properties, breast cancer cells were grown as mammospheres. GTCpFE, as well as dimethyl fumarate (DMF), prevented formation of MCF-7 mammospheres in a dose-dependent manner. Similar results were observed with other specific NFKB pathway inhibitors, such as IKK7 and BAY11-7082 (Bay). In addition, GTCpFE, as well as DMF, prevented mammosphere formation in other breast cancer cells lines without affecting survival in parental 2D attached culture. These findings indicate that both GTCpFE and DMF might attenuate self-renewal, anchorage-independent growth or both.

[00271] To determine if GTCpFE can affect the NFKB pathway in mammospheres, single cells were cultured in mammosphere conditions for 7 days with compound added for the last 6 hrs. GTCpFE, as well as DMF, attenuate increased p65 DNA binding in (FIG. 7), nuclear translocation levels, and expression of NFKB target genes (FIGS. 8A-8C) in mammospheres.

[00272] To validate that reduced MS growth is in fact an attenuation of CSC properties, CD44+/CD24- phenotype changes were tested for in MDA-MD-231, a cell line previously shown to contain a higher percentage of this phenotype given their mesenchymal character. It was found that both GTCpFE and DMF inhibit MDA-MB-231 mammosphere formation (FIGS. 9A-9C) in addition to significantly reducing the CD44+/CD24- percentage (FIG. 10). The reduced CD44+/CD24- population resulted in reduced subsequent mammosphere growth of primary and secondary generation.

[00273] The anti-CSCs properties, and as a result the anti-cancer effects of GTCpFE and DMF were tested in a xenograft model in nude mice. GTCpFE is more potent than DMF at inhibiting mammosphere growth, the CD44+/CD24- phenotype and xenograft tumor growth by potentially targeting phosphorylation of p65 (S536), shown to be inhibited at both 2D culture in an IKK/IkBa-dependent manner, and in mammopheres (FIG. 1 1).

[00274] Cell culture and mammosphere assay. MCF-7 cells, T47D and BT474 cells were grown in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS), nonessential amino acids, L-glutamine, insulin 0.06 ng/ml and antibiotics. All cells are maintained at 37 °C in a humid atmosphere of 5% CO2. The mammosphere assay is carried out as previously described with minor modifications. DMEM/F12 medium with 1% methyl cellulose is supplemented with B27 (Gibco), human recombinant EGF, hydrocortisone, insulin and antibiotics. Cells are trypsinized and dissociated to single cells via a cell strainer and plated into 96-W low-attachment plates at 400cells/well (MCF-7, T47D and MDA-MB- 231), 1000 cells/well (BT474) density. Compounds are added the following day and mammosphere are allowed to grow for 7 days before taking pictures at 4X via a microscope and mammospheres larger than 50μιη in diameter are manually counted. [00275] Quantitative PCR. After treatment, RNA was isolated using Trizol, reverse transcribed and analyzed by quantitative PCR (QPCR) using specific primers (sequences are available upon request). 36B4 served as an internal control.

[00276] Transient tranfections. MCF-7 cells were transiently transfected with reporter constructs for NFKB-RE (Clontech) along with the Renilla luciferase construct, pGL4.70 (Promega), using Lipofectamine 2000 (Invitrogen) in antibiotic- free Opti-MEM. Dual Luciferase assays (Promega) were carried out after 4h of treatment with TNFa.

[00277] MTS viability assay. Cell viability upon drug treatment at different concentrations was measured via the MTS assay (Promega). Cells were seeded in 96W plates and allowed to attach overnight. Following treatment, cells were harvested at 24, 48 and 72h by incubating cells for lh at 37C with MTS reagent according to manufacturer's guidelines.

[00278] RelA p65 DNA binding assay. NFKB p65 DNA binding assay (TransAM) was carried out according to manufacturer's instructions. Cells were pretreated with NFKB inhibitors for 2h, followed by TNFa stimulation for 15 or 30 min. Cells were harvested and nuclear extracts were collected according to manufacturer's instructions (TransAM kit).

[00279] Statistical Analysis. QPCR and reporter data were analyzed by two-way ANOVA followed by a post-hoc Bonferroni test. Significance for all statistical tests was set at P<0.05. Data shown are the mean +/- SEM from at least three independent determinations.

Modulation of Nrf2 Signaling

[00280] Comparison of NO-ASA and X-ASA analogues showed that QM-forming compounds in cell culture covalently modified Keapl, activated ARE, upregulated cytoprotective target genes, induced translocation of Nrf2 to the nucleus, and activated an oxidatively sensitive "NO-specific" fluorescent reporter. The central importance of QM in NO-ASA chemopreventive actions led to the design of compounds of the invention that on bioactivation yield only salicylic acid, ASA and QM.

[00281] Results indicate that the major mechanism of Nrf2 signaling by QM-releasing NO- ASA and X-ASA is through modification of Keap 1 consequent to release of the bioactive QM metabolite. Compounds p(ASA) ₂ (Example 36) and o(ASA) ₂ (Example 37) were designed and synthesized. These compounds are bioactivated by esterase to release only salicylates and a QM. Comparison was made to two ASA derivatives, BrCH^-ASA and NOCH2-ASA, that cannot release a QM, measuring activation of ARE in HepG2 cells (FIG. 12). Both /?(ASA) ₂ and o(ASA)2 showed high efficacy towards activation of Nrf2/ARE signaling. [00282] Experimental Procedures. All chemicals and reagents were purchased from Sigma- Aldrich (St. Louis, MO) unless stated otherwise. The Comet Assay Kit was purchased from Trevigen Inc. (Gaithersburg, MD). Taqman gene expression assay master mix and primers with FAM/MGB probes were purchased from Applied Biosystems (Foster City, CA).

R easy mini kit was purchased from Qiagen (Valencia, CA). Nrf2 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Fluorescein conjugated secondary antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). VECTASHIELD mounting media with DAPI was purchased from Vector Laboratories, Inc. (Burlingame, CA). GST-PI and GST-PI(CIOIA) were expressed from E. coli.

[00283] Cell Cultures. Human Hepatoma HepG2 cells were obtained from American Type Culture Collection (Manassas, VA). Hepa lclc7 murine Hepatoma cells were supplied by Dr. J.P.Whitlock, Jr. (Stanford University, Stanford, CA) and were cultured in a-MEM with 1% penicillin-streptomycin and 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA).

HepG2 cells stably transfected with ARE-luciferase reporter gene were kindly provided by Dr. A.N. Kong (Rutgers University, Piscataway, NJ) and cultured in F-12 medium with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% non-essential amino acids and insulin (0.2 mg/mL).

[00284] GSH Levels. The total cellular glutathione (GSH + GSSG) level in HepG2 cells was measured by an enzymatic recycling procedure with some modifications as described previously.

[00285] Total RNA isolation and qPCR. HepG2 cells at a density of 4 x 10 ⁶ cells/mL were plated in 100 mm culture plates and treated with DMSO vehicle and compounds for 4 h. Total RNA was extracted from cell pellets using the RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Total RNA (5 μg) was reverse transcribed using oligo(dT) ₁₂ _ ₁₈ (500 ug mL) _, dNTP (10 mM), and DEPC H ₂ 0, which was added to equal a volume of 12 μΕ. The mixture was heated at 65°C for 5 min and thereafter quickly chilled on ice. The resulting solution was collected by centrifugation and added to DTT (0.1 M) and 4 first-strand buffer (40 units^L), giving a total volume of 19 μί. The solution was heated at 42°C for 2 min before 1 μΐ _^ of Superscript II RT (200 units) was added. The reaction was incubated at 42°C for an additional 90 min. The resulting cDNA (2 μΕ) was used for realtime PCR quantification (7500 Real-Time PCR System). Taqman gene expression master mix, containing AmpliTaq Gold® DNA polymerase, and a GCLC primer with FAM/MGB probe (Applied Biosystems, Carlsbad, CA) was added to MicroAmp® Optical 96-well reaction plate Real-time quantitative PCR consisted of one cycle of 50°C for 2 min and 95°C for lO min and 40 cycles of 95°C for 15 sec and 60°C for 1 min. The fluorescence signal was measured during the last 30 sec of the annealing/extension phase. Data was analyzed using comparative CT (ΔΔΟχ) method, using actin as the endogenous control. The results are expressed as fold-difference in reference to actin control.

[00286] Nrf2 translocation. HepG2 cells at a density of 5xl0 ⁴ cells/mL were plated in 8- well Nunc™ chamber slides coated with poly-d-lysine. Cells were treated with compounds for 4 h, 18 h, or 24 h and incubated at 37°C. Media was removed and the cells were fixed with 4% paraformaldehyde for 10 min. Cells were then washed with PBS and 0.2% Triton X-100. Triton X-100 solution was removed after 10 min and slides were washed for an additional 10 min with PBS. Blocking was done with 10% normal goat serum added to each sample and placed on a shaker for 2 h. The cells were incubated with rabbit polyclonal anti- Nrf2 antibody overnight. Cells were incubated with fluorescein conjugated anti-rabbit IgG secondary antibody for 1 h and 4'-6-diamidino-2-phenylindole (DAPI) was added to the cells to detect nuclear staining. Imaging was performed with a Zeiss LSM 510 laser-scanning confocal microscope with the detector gain adjusted to eliminate the background

autofluorescence. The fluorescence signal from Nrf2 was monitored with a 488 nm argon/krypton laser and a 530 nm band pass filter. The DAPI nuclear staining signal was monitored with a 345 nm UV laser and 420 nm band pass filter. Images were analyzed using the analysis tool provided in the Zeiss biophysical software package and ImageJ.

[00287] NQO l activity assay. For cultured Hepa lclc7 liver cells, the induction of NQOl activity was assayed as described previously with minor modifications.

[00288] NA damage in Hepa lclc7 cells using Single-Cell Gel Electrophoresis Assay (Comet assay). This assay was performed according to the manufacturer's recommendations (Trevigen, Inc. Gaithersburg, MD) as described previously.

[00289] ARE-Luciferase Reporter Assay. HepG2-ARE-Luc cells were plated in 6-well plates at a density of 1 xlO ⁵ and luciferase activity, normalized to protein concentration, measured as described previously.

[00290] GST-P 1 and Keap 1 reactions with pNO-ASA or pBr-ASA. For the analysis of alkylation of GST-P 1 and Keapl Cys residues, GST-P 1 or Keapl (15 μΜ) was incubated with 30 μΜ substrate (pNO-ASA/ Br-ASA) in the presence of porcine liver esterase (PLE, 30 μg/mL) in phosphate buffer (40 mM, pH 7.4) for 30 min at 37 °C. The unreacted thiols were then blocked by incubating the protein samples with NEM (20 mM) in the presence of 5% SDS for 30 min at 55 °C with vortexing at 5 min intervals. Prepared samples were run on SDS gel, and the separated protein bands were stained with Coomasie Brilliant Blue. Finally, bands containing GST-PI or Kea l, determined by visual inspection, were excised, destained, and digested using trypsin or Lys-C enzymes according to the manufacturer's (Thermo Pierce, IL) instructions. The amino acid residues on either side of CyslOl in GST- PI, ArglOO and Lys l02, are cleavage sites for trypsin digestion, therefore, Lys-C digestion of GST-PI was used to obtain a detectable peptide containing CyslOl for these studies.

[00291] Mass spectroscopic analysis of digested proteins. The digested protein samples were analyzed with an Agilent 6310 ion trap mass spectrometer (Agilent Technologies, Santa Clara, CA) coupled to Agilent 1 100 series HPLC system with ESI. The samples were separated using a Hypersil BSD reversed-phase CI 8 column (30 x 2.1 mm, 3μιη, Thermo Scientific) with gradient 90/10 (v/v) water/methanol , 0.1% formic acid and acetonitrile, 0.1% formic acid mobile phase at 300 uL/min flow rate. The positive mode electrospray ionization method was utilized for all the LC -MS/MS analyses. For the quantitative analysis of QM modification of GST in the presence of free Cys, the peak area for the QM-modified Cys47 containing peptide fragment (residue 45-54: ASCLYGQLPK) was measured and normalized against the peak area of the adjacent peptide fragment (residue 55-70:

FQDGDLTLYQSNTILR) to quantitatively estimate the extent of QM modification of Cys47 with respect to cysteine concentration.

[00292] Protein digests were also analyzed using a hybrid linear ion trap FT-ICR mass spectrometer (LTQ-FT-ICR, Thermo Electron Corp., Bremen, Germany) equipped with a nanospray ESI source and nano-HPLC with autosampler (Dionex, Sunnyvale, CA). The trapping cartridge and the nano-column used for separation were Zorbax 300 SB-C18 (5 x 0.3 mm, 5 μιη, Agilent Technologies) and Zorbax 300 SB-C18 (150 mm x 75 μιη, 3.5 μιη, Agilent Technologies), respectively. The separations were carried with gradient elution (250 nL/min; 5% acetonitrile, 0.1 % formic acid to 95% acetonitrile, 0.1% formic acid). The RAW files were converted to mzXML files and they were searched by the MassMatrix

(http://www.massmatrix.net) search engine against the UniProt human database with NEM and QM as the variable modifications. The area of the QM-modified peptide peak was divided by the combined peak areas of the NEM- and QM-modified peptide peaks to estimate the percentage QM modifications. For these quantitative estimations the ionization properties of a given modified peptide are assumed to be similar, which given the small and very similar change in peptide mass caused by QM and NEM modification is reasonable.

[00293] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents. [00294] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.