NIFUROXAZIDE ANALOGS AND THERAPEUTIC USES THEREOF Background

Field

[0001] The present disclosure relates to the fields of chemistry and medicine. More particularly, the present disclosure relates to nifuroxazide analogs, compositions, their preparation, and their use as therapeutic agents. Description of the Related Art

[0002] Signal transduce and activator of transcription 3 (STAT3) is a transcription factor that mediates the expression of a variety of genes in response to cell stimuli and plays a key role in many cellular processes. Constitutive activation of the transcription factor STAT3 contributes to the pathogenesis of many cancers, including multiple myeloma, melanoma, colorectal cancer, and breast cancer. Since STAT3 is dispensable in most normal tissue, targeted inhibition of STAT3 is an attractive therapy for patients with these cancers.

[0003] Nifuroxazide is a broad spectrum nitrofuran antibiotic that is useful in the treatment of colitis and diarrhea in humans and animals. Nifuroxazide is an effective inhibitor of STAT3 function and has demonstrated unique antitumor activity. In particular, nifuroxazide decreases the viability of three breast cancer cell lines and induce apoptosis of cancer cells in a dose-dependent manner. Moreover, nifuroxazide demonstrated markedly blocked cancer cell migration and invasion, and reduced phosphorylated-STAT3(Tyr705), matrix metalloproteinase (MMP) MMP-2 and MMP-9 expression. It is believed that nifuroxazide preferentially targets tumor cell metabolism and moderates paracrine induction of STAT3.

[0004] Thus, it is desirable to develop small-molecules that are structurally related to nifuroxazide as potential targeted cancer therapeutics. Summary of the Disclosure

[0005] Some embodiments of the present application provide a compound of Formula I, including pharmaceutically acceptable salts thereof:

[0006] In some embodiments, represents either a single bond or a double bond.

[0007] In some embodiments, R ¹ is selected from the group consisting of unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S.

[0008] In some embodiments, a moiety indicated as substituted is substituted with one or more Q. In some embodiments, each Q is independently selected from the group consisting of C _1-4 alkyl, C _1-4 haloalkyl, C _3-6 cycloalkyl, C _3-10 heterocycloalkyl, C _6-10 aryl, C _6-10 arylalkyl, C _3-10 heteroaryl, C _3-10 heteroarylalkyl, halo, oxo, -OH, -O-(C _1-4 alkyl), -O-(C _1-4 haloalkyl), -SH, -S-(C _1-4 alkyl), -S-(C _1-4 haloalkyl), -S(O)2-(C _1-4 alkyl), -S(O)2-(C _1-4 haloalkyl), -S(O) ₂ NR ^a R ^a , -NR ^a R ^a , -NO ₂ -CN, -C(O)NR ^a R ^a , and–NR ^a C(O)R ^a . In some embodiments, each R ^a is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0009] In some embodiments, R ^A , R ^B , and R ^C are each independently selected from hydrogen and–NO ₂ , wherein one of R ^A , R ^B , and R ^C is–NO ₂ .

[0010] In some embodiments, R ¹ is not unsubstituted phenyl.

[0011] In some embodiments, R ¹ is not (Z)-5-((4-nitrocyclopenta-1,3-dien-1- yl)methylene)-3-phenylthiazolidine-2,4-dione.

[0012] In some embodiments, R ^A is–NO2. In some embodiments, R ^B is–NO2. In some embodiments, R ^C is–NO ₂ .

[0013] In some embodiments, represents a single bond. In some

embodiments, represents a double bond. [0014] In some embodiments, R ¹ is unsubstituted C _6-10 aryl. In some embodiments, R ¹ is substituted C6-10 aryl. In some embodiments, R ¹ is unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. In some embodiments, R ¹ is substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S.

[0015] In some embodiments of the present application the compound of Formula I has the structure depicted in Formula Ia [0016] In some embodiments, represents a single bond or a double bond.

[0017] In some embodiments, each Q is selected from the group consisting of C _1-4 alkyl, C _1-4 haloalkyl, C _3-6 cycloalkyl, C _3-10 heterocycloalkyl, C _6-10 aryl, C _6-10 arylalkyl, C _3-10 heteroaryl, C3-10 heteroarylalkyl, halo, oxo, -OH, -O-(C _1-4 alkyl), -O-(C _1-4 haloalkyl), -SH, -S- (C _1-4 alkyl), -S-(C _1-4 haloalkyl), -S(O) ₂ -(C _1-4 alkyl), -S(O) ₂ -(C _1-4 haloalkyl), -S(O) ₂ NR ^a R ^a , - NR ^a R ^a , -NO ₂ -CN, -C(O)NR ^a R ^a , and–NR ^a C(O)R ^a , wherein each R ^a is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0018] In some embodiments, n is 1, 2, 3, 4, or 5.

[0019] In some embodiments, represents a single bond. In some embodiments, represents a double bond.

[0020] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0021] In some embodiments of the present application the compound of Formula I has the structure depicted in Formula 1b [0022] In some embodiments, represents a single bond or a double bond.

[0023] In some embodiments, hAr is five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S.

[0024] In some embodiments, each Q is selected from the group consisting of C _1-4 alkyl, C _1-4 haloalkyl, C _3-6 cycloalkyl, C _3-10 heterocycloalkyl, C _6-10 aryl, C _6-10 arylalkyl, C _3-10 heteroaryl, C3-10 heteroarylalkyl, halo, oxo, -OH, -O-(C _1-4 alkyl), -O-(C _1-4 haloalkyl), -SH, -S- (C _1-4 alkyl), -S-(C _1-4 haloalkyl), -S(O) ₂ -(C _1-4 alkyl), -S(O) ₂ -(C _1-4 haloalkyl), -S(O) ₂ NR ^a R ^a , - NR ^a R ^a , -NO ₂ -CN, -C(O)NR ^a R ^a , and–NR ^a C(O)R ^a . In some embodiments, each R ^a is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

[0025] In some embodiments, m is 0, 1, 2, 3, 4, or 5.

[0026] In some embodiments, the pharmaceutically acceptable salt is an alkaline metal salt or an ammonium salt.

[0027] In certain embodiments disclosed herein, a pharmaceutical composition comprising a therapeutically effective amount of at least one compound having the structure of the Formula I, including pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients, carriers, diluents, or combination thereof.

[0028] Other embodiments disclosed herein include a method of ameliorating or treating a mammal having a disease disease or disorder comprising administering to the mammal a therapeutically effective amount of a compound of Formula I.

[0029] In some embodiments, the mammal is a human.

[0030] Other embodiments disclosed herein further comprising administering to the subject an additional medicament.

[0031] Other embodiments disclosed herein include additional medicaments selected from an antibacterial agent, an antifungal agent, an antiviral agent, an anti- inflammatory agent, or an anti-allergic agent.

[0032] Other embodiments disclosed herein include additional medicaments selected from Tocilizumab or Siltuximab. Brief Description of the Drawings

[0033] FIGURE 1 shows the effect of drug-induced transient mitochondria-SOX induction in MDA-MB-231 cells.

[0034] FIGURE 2A shows the effect of Compound 1 on downregulation of pSTAT3 ^Y705 in MCF-7 tumor cells.

[0035] FIGURE 2B shows the effect of Compound 1 on downregulation of pSTAT3 ^Y705 in BT-474 tumor cells.

[0036] FIGURE 2C shows the effect of Compound 1 on downregulation of pSTAT3 ^Y705 in SK-BR03 tumor cells.

[0037] FIGURE 3 shows the effect of Compound 1 on the inhibition of pSTAT1 ^Y701 in HeLa cells.

[0038] FIGURE 4A shows the effect of Compound 1 in CT26 athymic nude Balb/c (immune deficient) mice.

[0039] FIGURE 4B shows the effect of anti-PD-1 monoclonal antibody and anti- CTLA-4 monoclonal antibody in CT26 athymic nude Balb/c (immune deficient) mice.

[0040] FIGURE 4C shows the effect of the combination of Compound 1 and either anti-PD-1 monoclonal antibody or anti-CTLA-4 monoclonal antibody in CT26 athymic nude Balb/c (immune deficient) mice.

[0041] FIGURE 5 shows the effect of Compound 1 on tumor growth inhibition in CT26 Balb/c (immune competent) mice.

[0042] FIGURE 6 shows the effect of the combination of Compound 1 and either anti-PD-1 monoclonal antibody or anti-CTLA-4 monoclonal antibody on tumor growth inhibition in CT26 Balb/c (immune competent) mice. Detailed Description of the Preferred Embodiment

[0043] In some embodiments, nifuroxazide analogs are provided that act as an anti-proliferative agent. Various embodiments of these compounds include compounds having the structure of Formula I as described above or pharmaceutically acceptable salts thereof. [0044] In some embodiments, prodrugs, metabolites, stereoisomers, hydrates, solvates, polymorphs, and pharmaceutically acceptable salts of the compounds disclosed herein are provided.

[0045] The term“prodrug,” as used herein, refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. An example, without limitation, of a prodrug would be a compound which is administered as an ester (the“prodrug”) to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water-solubility is beneficial. A further example of a prodrug might be a short peptide (polyaminoacid) bonded to an acid group where the peptide is metabolized to reveal the active moiety. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in Design of Prodrugs, (ed. H. Bundgaard, Elsevier, 1985), which is hereby incorporated herein by reference in its entirety.

[0046] The term“pro-drug ester,” as used herein, refers to derivatives of the compounds disclosed herein formed by the addition of any of several ester-forming groups that are hydrolyzed under physiological conditions. Examples of pro-drug ester groups include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, as well as other such groups known in the art, including a (5-R-2-oxo-1,3-dioxolen-4-yl)methyl group. Other examples of pro-drug ester groups can be found in, for example, T. Higuchi and V. Stella, in "Pro-drugs as Novel Delivery Systems", Vol.14, A.C.S. Symposium Series, American Chemical Society (1975); and "Bioreversible Carriers in Drug Design: Theory and Application", edited by E. B. Roche, Pergamon Press: New York, 14-21 (1987) (providing examples of esters useful as prodrugs for compounds containing carboxyl groups). Each of the above-mentioned references is herein incorporated by reference in their entirety.

[0047] Metabolites of the compounds disclosed herein include active species that are produced upon introduction of the compounds into the biological milieu. [0048] Where the compounds disclosed herein have at least one chiral center, they may exist as a racemate or as enantiomers. It should be noted that all such isomers and mixtures thereof are included in the scope of the present disclosure. Furthermore, some of the crystalline forms for the compounds disclosed herein may exist as polymorphs. Such polymorphs are included in one embodiment of the present disclosure. In addition, some of the compounds of the present disclosure may form solvates with water (i.e., hydrates) or common organic solvents. Such solvates are included in one embodiment of the present disclosure.

[0049] The term“pharmaceutically acceptable salt,” as used herein, refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutical salts can be obtained by reacting a compound with inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, phosphoric acid and the like. Pharmaceutical salts can also be obtained by reacting a compound with an organic acid such as aliphatic or aromatic carboxylic or sulfonic acids, for example acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic, p-toluensulfonic, salicylic or naphthalenesulfonic acid. Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine, and salts with amino acids such as arginine, lysine, and the like.

[0050] If the manufacture of pharmaceutical formulations involves intimate mixing of the pharmaceutical excipients and the active ingredient in its salt form, then it may be desirable to use pharmaceutical excipients which are non-basic, that is, either acidic or neutral excipients.

[0051] In various embodiments, the compounds disclosed herein can be used alone, in combination with other compounds disclosed herein, or in combination with one or more other agents active in the therapeutic areas described herein. [0052] The term“halogen atom,” as used herein, means any one of the radio- stable atoms of column 7 of the Periodic Table of the Elements, e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.

[0053] The term“ester,” as used herein, refers to a chemical moiety with formula -(R)n-COOR’, where R and R’ are independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), and where n is 0 or 1.

[0054] The term“amide,” as used herein, refers to a chemical moiety with formula -(R)n-C(O)NHR’ or -(R)n-NHC(O)R’, where R and R’ are independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), and where n is 0 or 1. An amide may be an amino acid or a peptide molecule attached to a molecule of the present disclosure, thereby forming a prodrug.

[0055] Any amine, hydroxyl, or carboxyl side chain on the compounds disclosed herein can be esterified or amidified. The procedures and specific groups to be used to achieve this end are known to those of skill in the art and can readily be found in reference sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3 ^rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein in its entirety.

[0056] The term“aromatic,” as used herein, refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes both carbocyclic aryl (e.g., phenyl) and heterocyclic aryl groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups. The term“carbocyclic” refers to a compound which contains one or more covalently closed ring structures, and that the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocyclic from heterocyclic rings in which the ring backbone contains at least one atom which is different from carbon. The term“heteroaromatic” refers to an aromatic group which contains at least one heterocyclic ring.

[0057] As used herein,“alkyl” refers to a straight or branched hydrocarbon chain fully saturated (no double or triple bonds) hydrocarbon group. The alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as“1 to 20” refers to each integer in the given range; e.g.,“1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term“alkyl” where no numerical range is designated). The alkyl group may also be a medium size alkyl having 1 to 10 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 5 carbon atoms. The alkyl group of the compounds may be designated as“C ₁ -C ₄ alkyl” or similar designations. By way of example only,“C1-C4 alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, and the like.

[0058] The alkyl group may be substituted or unsubstituted. When substituted, the substituent group(s) is(are) one or more group(s) individually and independently selected from alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, ester, mercapto, alkylthio, arylthio, cyano, halogen, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof. Wherever a substituent is described as being“optionally substituted” that substituent may be substituted with one of the above substituents.

[0059] As used herein,“alkenyl” refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more double bonds. An alkenyl group may be unsubstituted or substituted. When substituted, the substituent(s) may be selected from the same groups disclosed above with regard to alkyl group substitution. The alkenyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term“alkenyl” where no numerical range is designated. The alkenyl group may also be a medium size alkenyl having 2 to 9 carbon atoms. The alkenyl group could also be a lower alkenyl having 2 to 4 carbon atoms. The alkenyl group of the compounds may be designated as“C2-4 alkenyl” or similar designations. By way of example only,“C2-4 alkenyl” indicates that there are two to four carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl- ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups include, but are in no way limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl, and the like.

[0060] As used herein,“alkynyl” refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more triple bonds. An alkynyl group may be unsubstituted or substituted. When substituted, the substituent(s) may be selected from the same groups disclosed above with regard to alkyl group substitution. The alkynyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term“alkynyl” where no numerical range is designated. The alkynyl group may also be a medium size alkynyl having 2 to 9 carbon atoms. The alkynyl group could also be a lower alkynyl having 2 to 4 carbon atoms. The alkynyl group of the compounds may be designated as“C2-4 alkynyl” or similar designations. By way of example only,“C2-4 alkynyl” indicates that there are two to four carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn- 4-yl, and 2-butynyl. Typical alkynyl groups include, but are in no way limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like.

[0061] As used herein,“heteroalkyl” refers to a straight or branched hydrocarbon chain containing one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the chain backbone. The heteroalkyl group may have 1 to 20 carbon atoms although the present definition also covers the occurrence of the term“heteroalkyl” where no numerical range is designated. The heteroalkyl group may also be a medium size heteroalkyl having 1 to 9 carbon atoms. The heteroalkyl group could also be a lower heteroalkyl having 1 to 4 carbon atoms. The heteroalkyl group of the compounds may be designated as“C _1-4 heteroalkyl” or similar designations. The heteroalkyl group may contain one or more heteroatoms. By way of example only,“C _1-4 heteroalkyl” indicates that there are one to four carbon atoms in the heteroalkyl chain and additionally one or more heteroatoms in the backbone of the chain.

[0062] As used herein,“aryl” refers to a carbocyclic (all carbon) ring or two or more fused rings (rings that share two adjacent carbon atoms) that have a fully delocalized pi- electron system. Examples of aryl groups include, but are not limited to, benzene, naphthalene and azulene. An aryl group may be substituted or unsubstituted. When substituted, hydrogen atoms are replaced by substituent group(s) that is(are) one or more group(s) independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, ester, mercapto, alkylthio, arylthio, cyano, halogen, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C- carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof. When substituted, substituents on an aryl group may form a non-aromatic ring fused to the aryl group, including a cycloalkyl, cycloalkenyl, cycloalkynyl, and heterocyclyl.

[0063] As used herein,“heteroaryl” refers to a monocyclic or multicyclic aromatic ring system (a ring system with fully delocalized pi-electron system), one or two or more fused rings that contain(s) one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur. Examples of heteroaryl rings include, but are not limited to, furan, thiophene, phthalazine, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole, isothiazole, triazole, thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine and triazine. A heteroaryl group may be substituted or unsubstituted. When substituted, hydrogen atoms are replaced by substituent group(s) that is(are) one or more group(s) independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, ester, mercapto, alkylthio, arylthio, cyano, halogen, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C- carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof. When substituted, substituents on a heteroayl group may form a non-aromatic ring fused to the aryl group, including a cycloalkyl, cycloalkenyl, cycloalkynyl, and heterocyclyl.

[0064] As used herein, an“aralkyl” or“arylalkyl” refers to an aryl group connected, as a substituent, via an alkylene group. The alkylene and aryl group of an aralkyl may be substituted or unsubstituted. Examples include but are not limited to benzyl, substituted benzyl, 2-phenylethyl, 3-phenylpropyl, and naphtylalkyl. In some cases, the alkylene group is a lower alkylene group.

[0065] As used herein, a“heteroaralkyl” or“heteroarylalkyl” is heteroaryl group connected, as a substituent, via an alkylene group. The alkylene and heteroaryl group of heteroaralkyl may be substituted or unsubstituted. Examples include but are not limited to 2- thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazollylalkyl, and imidazolylalkyl, and their substituted as well as benzo-fused analogs. In some cases, the alkylene group is a lower alkylene group.

[0066] As used herein, a“alkylene” refers to a branched, or straight chain fully saturated di-radical chemical group containing only carbon and hydrogenthat is attached to the rest of the molecule via two points of attachment (i.e., an alkanediyl). The alkylene group may have 1 to 20 carbon atoms, although the present definition also covers the occurrence of the term alkylene where no numerical range is designated. The alkylene group may also be a medium size alkylene having 1 to 9 carbon atoms. The alkylene group could also be a lower alkylene having 1 to 4 carbon atoms. The alkylene group may be designated as“C _1-4 alkylene” or similar designations. By way of example only,“C _1-4 alkylene” indicates that there are one to four carbon atoms in the alkylene chain, i.e., the alkylene chain is selected from the group consisting of methylene, ethylene, ethan-1,1-diyl, propylene, propan-1,1-diyl, propan-2,2-diyl, 1-methyl-ethylene, butylene, butan-1,1-diyl, butan-2,2-diyl, 2-methyl- propan-1,1-diyl, 1-methyl-propylene, 2-methyl-propylene, 1,1-dimethyl-ethylene, 1,2- dimethyl-ethylene, and 1-ethyl-ethylene. [0067] As used herein,“alkenylene” refers to a straight or branched chain di- radical chemical group containing only carbon and hydrogen and containing at least one carbon-carbon double bond that is attached to the rest of the molecule via two points of attachment. The alkenylene group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term alkenylene where no numerical range is designated. The alkenylene group may also be a medium size alkenylene having 2 to 9 carbon atoms. The alkenylene group could also be a lower alkenylene having 2 to 4 carbon atoms. The alkenylene group may be designated as“C2-4 alkenylene” or similar designations. By way of example only,“C _2-4 alkenylene” indicates that there are two to four carbon atoms in the alkenylene chain, i.e., the alkenylene chain is selected from the group consisting of ethenylene, ethen-1,1-diyl, propenylene, propen-1,1-diyl, prop-2-en-1,1-diyl, 1-methyl- ethenylene, but-1-enylene, but-2-enylene, but-1,3-dienylene, buten-1,1-diyl, but-1,3-dien-1,1- diyl, but-2-en-1,1-diyl, but-3-en-1,1-diyl, 1-methyl-prop-2-en-1,1-diyl, 2-methyl-prop-2-en- 1,1-diyl, 1-ethyl-ethenylene, 1,2-dimethyl-ethenylene, 1-methyl-propenylene, 2-methyl- propenylene, 3-methyl-propenylene, 2-methyl-propen-1,1-diyl, and 2,2-dimethyl-ethen-1,1- diyl.

[0068] As used herein,“alkylidene” refers to a divalent group, such as =CR’R’’, which is attached to one carbon of another group, forming a double bond, alkylidene groups include, but are not limited to, methylidene (=CH2) and ethylidene (=CHCH3). As used herein,“arylalkylidene” refers to an alkylidene group in which either R’ and R’’ is an aryl group. An alkylidene group may be substituted or unsubstituted.

[0069] As used herein,“alkoxy” refers to the formula–OR wherein R is an alkyl is defined as above, e.g. methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n- butoxy, iso-butoxy, sec-butoxy, tert-butoxy, amoxy, tert-amoxy and the like. An alkoxy may be substituted or unsubstituted.

[0070] As used herein,“alkylthio” refers to the formula–SR wherein R is an alkyl is defined as above, e.g. methylmercapto, ethylmercapto, n-propylmercapto, 1- methylethylmercapto (isopropylmercapto), n-butylmercapto, iso-butylmercapto, sec- butylmercapto, tert-butylmercapto, and the like. An alkylthio may be substituted or unsubstituted. [0071] As used herein, “aryloxy” and “arylthio” refers to RO- and RS-, respectively, in which R is an aryl, such as but not limited to phenyl. Both an aryloxyl and arylthio may be substituted or unsubstituted.

[0072] As used herein,“acyl” refers to–C(=O)R, wherein R is hydrogen, C _1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein. Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, and acryl.

[0073] As used herein,“cycloalkyl” refers to a completely saturated (no double bonds) mono- or multi- cyclic hydrocarbon ring system. When composed of two or more rings, the rings may be joined together in a fused, bridged or spiro-connected fashion. Cycloalkyl groups may range from C ₃ to C ₁₀ , in other embodiments it may range from C ₃ to C6. A cycloalkyl group may be unsubstituted or substituted. Typical cycloalkyl groups include, but are in no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. If substituted, the substituent(s) may be an alkyl or selected from those indicated above with regard to substitution of an alkyl group unless otherwise indicated. When substituted, substituents on a cycloalkyl group may form an aromatic ring fused to the cycloalkyl group, including an aryl and a heteroaryl.

[0074] As used herein,“cycloalkenyl” refers to a cycloalkyl group that contains one or more double bonds in the ring although, if there is more than one, they cannot form a fully delocalized pi-electron system in the ring (otherwise the group would be“aryl,” as defined herein). When composed of two or more rings, the rings may be connetected together in a fused, bridged or spiro-connected fashion. A cycloalkenyl group may be unsubstituted or substituted. When substituted, the substituent(s) may be an alkyl or selected from the groups disclosed above with regard to alkyl group substitution unless otherwise indicated. When substituted, substituents on a cycloalkenyl group may form an aromatic ring fused to the cycloalkenyl group, including an aryl and a heteroaryl.

[0075] As used herein,“cycloalkynyl” refers to a cycloalkyl group that contains one or more triple bonds in the ring. When composed of two or more rings, the rings may be joined together in a fused, bridged or spiro-connected fashion. A cycloalkynyl group may be unsubstituted or substituted. When substituted, the substituent(s) may be an alkyl or selected from the groups disclosed above with regard to alkyl group substitution unless otherwise indicated. When substituted, substituents on a cycloalkynyl group may form an aromatic ring fused to the cycloalkynyl group, including an aryl and a heteroaryl.

[0076] As used herein,“heteroalicyclic” or“heteroalicyclyl” refers to a stable 3- to 18 membered ring which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. The“heteroalicyclic” or “heteroalicyclyl” may be monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may be joined together in a fused, bridged or spiro-connected fashion; and the nitrogen, carbon and sulfur atoms in the“heteroalicyclic” or“heteroalicyclyl” may be optionally oxidized; the nitrogen may be optionally quaternized; and the rings may also contain one or more double bonds provided that they do not form a fully delocalized pi-electron system throughout all the rings. Heteroalicyclyl groups may be unsubstituted or substituted. When substituted, the substituent(s) may be one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, ester, mercapto, alkylthio, arylthio, cyano, halogen, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof. Examples of such“heteroalicyclic” or“heteroalicyclyl” include but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, morpholinyl, oxiranyl, piperidinyl N-Oxide, piperidinyl, piperazinyl, pyrrolidinyl, 4-piperidonyl, pyrazolidinyl, 2-oxopyrrolidinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, and thiamorpholinyl sulfone. When substituted, substituents on a heteroalicyclyl group may form an aromatic ring fused to the heteroalicyclyl group, including an aryl and a heteroaryl.

[0077] As used herein, the term“(cycloalkenyl)alkyl” refers to a cycloalkenyl group connected, as a substituent, via an alkylene group. The alkylene and cycloalkenyl of a (cycloalkenyl)alkyl may be substituted or unsubstituted. In some cases, the alkylene group is a lower alkylene group.

[0078] As used herein, the term“(cycloalkynyl)alkyl” to a cycloalkynyl group connected, as a substituent, via an alkylene group. The alkylene and cycloalkynyl of a (cycloalkynyl)alkyl may be substituted or unsubstituted. In some cases, the alkylene group is a lower alkylene group.

[0079] As used herein, the term“O-carboxy” refers to a“RC(=O)O-” group in which R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl, as defined herein. An O-carboxy may be substituted or unsubstituted.

[0080] As used herein, the term“C-carboxy” refers to a“-C(=O)R” group in which R can be the same as defined with respect to O-carboxy. A C-carboxy may be substituted or unsubstituted.

[0081] As used herein, the term“trihalomethanesulfonyl” refers to an“X ₃ CSO ₂ -“ group wherein X is a halogen.

[0082] As used herein, the term“cyano” refers to a“-CN” group.

[0083] As used herein, the term“cyanato” refers to an“-OCN” group.

[0084] As used herein, the term“isocyanato” refers to a“-NCO” group.

[0085] As used herein, the term“thiocyanato” refers to a“-SCN” group.

[0086] As used herein, the term“isothiocyanato” refers to an“-NCS” group.

[0087] As used herein, the term“sulfinyl” refers to a“-S(=O)-R” group in which R can be the same as defined with respect to O-carboxy. A sulfinyl may be substituted or unsubstituted.

[0088] As used herein, the term“sulfonyl” refers to an“SO ₂ R” group in which R can be the same as defined with respect to O-carboxy. A sulfonyl may be substituted or unsubstituted.

[0089] As used herein, the term“S-sulfonamido” refers to a“-SO2NRARB” group in which R _A and R _B can be the same as defined with respect to O-carboxy. An S-sulfonamido may be substituted or unsubstituted. [0090] As used herein, the term“N-sulfonamido” refers to a“RSO ₂ N(R _A )-” group in which R and RA can be the same as defined with respect to O-carboxy. A sulfonyl may be substituted or unsubstituted.

[0091] As used herein, the term “trihalomethanesulfonamido” refers to an “X3CSO2N(R)-“ group with X as halogen and R can be the same as defined with respect to O-carboxy. A trihalomethanesulfonamido may be substituted or unsubstituted.

[0092] As used herein, the term“O-carbamyl” refers to a“-OC(=O)NR _A R _B ” group in which R _A and R _B can be the same as defined with respect to O-carboxy. An O-carbamyl may be substituted or unsubstituted.

[0093] As used herein, the term“N-carbamyl” refers to an“ROC(=O)NRA -“ group in which R and R _A can be the same as defined with respect to O-carboxy. An N-carbamyl may be substituted or unsubstituted.

[0094] As used herein, the term“O-thiocarbamyl” refers to a“-OC(=S)-NRARB” group in which RA and RB can be the same as defined with respect to O-carboxy. An O-thiocarbamyl may be substituted or unsubstituted.

[0095] As used herein, the term“N-thiocarbamyl” refers to an“ROC(=S)NRA-“ group in which R and RA can be the same as defined with respect to O-carboxy. An N-thiocarbamyl may be substituted or unsubstituted.

[0096] As used herein, the term“C-amido” refers to a“-C(=O)NR _A R _B ” group in which RA and RB can be the same as defined with respect to O-carboxy. A C-amido may be substituted or unsubstituted.

[0097] As used herein, the term“N-amido” refers to a“RC(=O)NR _A -“ group in which R and RA can be the same as defined with respect to O-carboxy. An N-amido may be substituted or unsubstituted.

[0098] As used herein, the term“amino” refers to a“-NRARB” group in which RA and R _B are each independently selected from hydrogen, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

[0099] As used herein, the term“aminoalkyl” refers to an amino group connected via an alkylene group. [0100] As used herein, the term“ester” refers to a“–C(=O)OR” group in which R can be the same as defined with respect to O-carboxy. An ester may be substituted or unsubstituted.

[0101] As used herein, the term lower aminoalkyl refers to an amino group connected via a lower alkylene group. A lower aminoalkyl may be substituted or unsubstituted.

[0102] As used herein, the term lower alkoxyalkyl refers to an alkoxy group connected via a lower alkylene group. A lower alkoxyalkyl may be substituted or unsubstituted.

[0103] As used herein, the term“acetyl” refers to a -C(=O)CH3, group.

[0104] As used herein, the term“trihalomethanesulfonyl” refers to a X3CS(=O)2- group where X is a halogen.

[0105] As used herein, the term“O-carbamyl” refers to a -OC(=O)-NR, in which R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl, as defined herein. An O- carbamyl can be substituted or unsubstituted.

[0106] As used herein, the term“N-carbamyl” refers to a ROC(=O)NH- group, in which R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl, as defined herein. An N- carbamyl can be substituted or unsubstituted.

[0107] As used herein, the term“O-thiocarbamyl” refers to a -OC(=S)-NR, in which R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl, as defined herein. An O- thiocarbamyl can be substituted or unsubstituted.

[0108] As used herein, the term“N-thiocarbamyl” refers to an ROC(=S)NH- group, in which R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl, as defined herein. An N-thiocarbamyl can be substituted or unsubstituted.

[0109] As used herein, the term“perhaloalkyl” refers to an alkyl group where all of the hydrogen atoms are replaced by halogen atoms. [0110] As used herein, the term“halogen” or“halo,” refer to any one of the radio- stable atoms of column 7 of the Periodic Table of the Elements, e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.

[0111] As used herein, the term“carbocyclyl” refers to a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system backbone. When the carbocyclyl is a ring system, two or more rings may be joined together in a fused, bridged or spiro-connected fashion. Carbocyclyls may have any degree of saturation provided that at least one ring in a ring system is not aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20 carbon atoms, although the present definition also covers the occurrence of the term“carbocyclyl” where no numerical range is designated. The carbocyclyl group may also be a medium size carbocyclyl having 3 to 10 carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3 to 6 carbon atoms. The carbocyclyl group may be designated as“C _3-6 carbocyclyl” or similar designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

[0112] As used herein, the term“(cycloalkyl)alkyl” refers to a cycloalkyl group connected, as a substituent, via an alkylene group. The alkylene and cycloalkyl of a (cycloalkyl)alkyl may be substituted or unsubstituted. Examples include but are not limited cyclopropylmethyl, cyclobutylmethyl, cyclopropylethyl, cyclopropylbutyl, cyclobutylethyl, cyclopropylisopropyl, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, cycloheptylmethyl, and the like. In some cases, the alkylene group is a lower alkylene group.

[0113] As used herein, the term“cycloalkyl” refers to a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0114] As used herein, the term“cycloalkenyl” means a carbocyclyl ring or ring system having at least one double bond, wherein no ring in the ring system is aromatic. An example is cyclohexenyl.

[0115] As used herein, the term“heterocyclyl” refers to three-, four-, five-, six-, seven-, and eight- or more membered rings wherein carbon atoms together with from 1 to 3 heteroatoms constitute said ring. A heterocyclyl can optionally contain one or more unsaturated bonds situated in such a way, however, that an aromatic pi-electron system does not arise. The heteroatoms are independently selected from oxygen, sulfur, and nitrogen.

[0116] As used herein, the term heterocyclyl can further contain one or more carbonyl or thiocarbonyl functionalities, so as to make the definition include oxo-systems and thio-systems such as lactams, lactones, cyclic imides, cyclic thioimides, cyclic carbamates, and the like.

[0117] As used herein,“heterocyclyl” refers to a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. Heterocyclyls may be joined together in a fused, bridged or spiro-connected fashion. Heterocyclyls may have any degree of saturation provided that at least one ring in the ring system is not aromatic. The heteroatom(s) may be present in either a non-aromatic or aromatic ring in the ring system. The heterocyclyl group may have 3 to 20 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term“heterocyclyl” where no numerical range is designated. The heterocyclyl group may also be a medium size heterocyclyl having 3 to 10 ring members. The heterocyclyl group could also be a heterocyclyl having 3 to 6 ring members. The heterocyclyl group may be designated as“3-6 membered heterocyclyl” or similar designations. In preferred six membered monocyclic heterocyclyls, the heteroatom(s) are selected from one up to three of O, N or S, and in preferred five membered monocyclic heterocyclyls, the heteroatom(s) are selected from one or two heteroatoms selected from O, N, or S. Examples of heterocyclyl rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4- dioxanyl, 1,3-oxathianyl, 1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

[0118] As used herein, the term“(heterocyclyl)alkyl” refers to a heterocyclyl group connected, as a substituent, via an alkylene group. Examples include, but are not limited to, imidazolinylmethyl and indolinylethyl.

[0119] The terms“purified,”“substantially purified,” and“isolated” as used herein, refer to compounds disclosed herein being free of other, dissimilar compounds with which the compounds of the disclosure are normally associated in their natural state, so that the compounds of the disclosure comprise at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample.

[0120] As used herein, a substituted group is derived from the unsubstituted parent group in which there has been an exchange of one or more hydrogen atoms for another atom or group. Unless otherwise indicated, when a group is deemed to be“substituted,” it is meant that the group is substituted with one or more substituents independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl, C ₁ -C ₆ heteroalkyl, C ₃ -C ₇ carbocyclyl (optionally substituted with halo, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl, and C ₁ -C ₆ haloalkoxy), C ₃ - C ₇ -carbocyclyl-C ₁ -C ₆ -alkyl (optionally substituted with halo, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ - C ₆ haloalkyl, and C ₁ -C ₆ haloalkoxy), 5-10 membered heterocyclyl (optionally substituted with halo, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl, and C ₁ -C ₆ haloalkoxy), 5-10 membered heterocyclyl-C ₁ -C ₆ -alkyl (optionally substituted with halo, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl, and C ₁ -C ₆ haloalkoxy), aryl (optionally substituted with halo, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl, and C ₁ -C ₆ haloalkoxy), aryl(C ₁ -C ₆ )alkyl (optionally substituted with halo, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl, and C ₁ -C ₆ haloalkoxy), 5- 10 membered heteroaryl (optionally substituted with halo, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl, and C ₁ -C ₆ haloalkoxy), 5-10 membered heteroaryl(C ₁ -C ₆ )alkyl (optionally substituted with halo, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl, and C ₁ -C ₆ haloalkoxy), halo, cyano, hydroxy, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkoxy(C ₁ -C ₆ )alkyl (i.e., ether), aryloxy, sulfhydryl (mercapto), halo(C ₁ -C ₆ )alkyl (e.g.,–CF ₃ ), halo(C ₁ -C ₆ )alkoxy (e.g.,–OCF ₃ ), C ₁ -C ₆ alkylthio, arylthio, amino, amino(C ₁ -C ₆ )alkyl, nitro, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C- carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl, and oxo (=O). Wherever a group is described as“optionally substituted” that group can be substituted with the above substituents.

[0121] In some embodiments, substituted group(s) is (are) substituted with one or more substituent(s) individually and independently selected from C1-C4 alkyl, amino, hydroxy, and halogen.

[0122] It is to be understood that certain radical naming conventions can include either a mono-radical or a di-radical, depending on the context. For example, where a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a di-radical. For example, a substituent identified as alkyl that requires two points of attachment includes di-radicals such as–CH2–,–CH2CH2–,–CH2CH(CH3)CH2–, and the like. Other radical naming conventions clearly indicate that the radical is a di-radical such as“alkylene” or“alkenylene.”

[0123] Unless otherwise indicated, when a substituent is deemed to be“optionally substituted,” it is meant that the substituent” is a group that may be substituted with one or more group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxyl, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof. The protecting groups that may form the protective derivatives of the above substituents are known to those of skill in the art and may be found in references such as Greene and Wuts, above.

[0124] The term“agent” or“test agent,” as used herein includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, peptide or mimetic, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms“agent”,“substance”, and“compound” are used interchangeably herein. [0125] The term“analog” is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved characteristics (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.

[0126] The term“mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rats and mice but also includes many other species.

[0127] The term“microbial infection,” as used herein, refers to the invasion of the host organism, whether the organism is a vertebrate, invertebrate, fish, plant, bird, or mammal, by pathogenic microbes. This includes the excessive growth of microbes that are normally present in or on the body of a mammal or other organism. More generally, a microbial infection can be any situation in which the presence of a microbial population(s) is damaging to a host mammal. Thus, a mammal is“suffering” from a microbial infection when excessive numbers of a microbial population are present in or on a mammal’s body, or when the effects of the presence of a microbial population(s) is damaging the cells or other tissue of a mammal. Specifically, this description applies to a bacterial infection. Note that the compounds of preferred embodiments are also useful in treating microbial growth or contamination of cell cultures or other media, or inanimate surfaces or objects, and nothing herein should limit the preferred embodiments only to treatment of higher organisms, except when explicitly so specified in the claims.

[0128] The term “pharmaceutically acceptable carrier” or“pharmaceutically acceptable excipient,” as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. In addition, various adjuvants such as are commonly used in the art may be included. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman’s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety.

[0129] The term“subject” as used herein, refers to a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.

[0130] The term“effective amount” or a“therapeutically effective amount” as used herein, refers to an amount of a therapeutic agent that is effective to relieve, to some extent, or to reduce the likelihood of onset of, one or more of the symptoms of a disease or condition, and includes curing a disease or condition.“Curing” means that the symptoms of a disease or condition are eliminated; however, certain long-term or permanent effects may exist even after a cure is obtained (such as extensive tissue damage).

[0131] The term“treat,”“treatment,” or“treating,” as used herein, refers to administering a pharmaceutical composition for prophylactic and/or therapeutic purposes. The term“prophylactic treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term “therapeutic treatment” refers to administering treatment to a

Compounds

[0132] Some embodiments of the present application generally relate to a compound of Formula I or a pharmaceutically acceptable salt thereof: [0133] wherein: represents a single bond or a double bond; R ¹ is selected from the group consisting of unsubstituted C6-10 aryl; substituted C6-10 aryl; unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; and substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S; wherein a moiety indicated as substituted is substituted with one or more Q, wherein Q is selected from the group consisting of C _1-4 alkyl, C _1-4 haloalkyl, C _3-6 cycloalkyl, C _3-10 heterocycloalkyl, C _6-10 aryl, C _6-10 arylalkyl, C _3-10 heteroaryl, C3-10 heteroarylalkyl, halo, oxo, -OH, -O-(C _1-4 alkyl), -O-(C _1-4 haloalkyl), -SH, -S-(C _1-4 alkyl), -S-(C _1-4 haloalkyl), -S(O)2-(C _1-4 alkyl), -S(O)2-(C _1-4 haloalkyl), -S(O) ₂ NR ^a R ^a , -NR ^a R ^a , -NO ₂ -CN, -C(O)NR ^a R ^a , and–NR ^a C(O)R ^a , wherein each R ^a is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; R ^A , R ^B , and R ^C are each independently selected from hydrogen and–NO2, wherein one of R ^A , R ^B , and R ^C is–NO2;

[0134] In some embodiments, when R ^A is–NO2, R ¹ is not unsubstituted phenyl.

[0135] In some embodiments, the compound of Formula (I) is not

[0136] In some embodiments the thiazolidinedione moiety can be replaced with a heterocyclic moiety. In some embodiments, the thiazolidinedione moiety can be replaced with five-membered heterocyclic moiety. For example, the thiazolidinedione moiety can be

replaced with moiety selected from, but not limited to, , ,

[0137] In some embodiments, the nitrofuran moiety (i.e. , , wherein one

of R ^A , R ^B , and R ^C is–NO ₂ ) can be replaced with a nitrothiophenyl moiety (

wherein one of R ^A , R ^B , and R ^C is–NO ₂ ).

[0138] In some embodiments, R ^A can be–NO2. In some embodiments, R ^B can be –NO2. In some embodiments, R ^C can be–NO2.

[0139] In some embodiments, represents a single bond. In some embodiments, represents a double bond.

[0140] In some embodiments, R ¹ can be unsubstituted C _6-10 aryl or substituted C _6- 10 aryl. Examples of suitable C6-10 aryl groups include, but are not limited to, phenyl and naphthyl.

[0141] In some embodiments, R ¹ can be unsubstituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S or substituted five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S. Examples of five- to ten-membered heteroaryl having 1-4 atoms selected from the group consisting of O, N, and S include, but are not limited to, furanyl, thiophenyl, pyrrolyl, thiazolyl, imidazolyl, pyrazolyl, oxazolyl, triazolyl, tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, quinolinyl, indazolyl, benzothiazolyl, benzimidazolyl, and quinazolinyl.

[0142] In some embodiments, the moieties indicated as substituted can be substituted with one or more Q. In some embodiments, the moieties indicated as substituted can be substituted with one, two, three, four, or five Q. In some embodiments Q can be C _1-4 alkyl, C _1-4 haloalkyl, C _3-6 cycloalkyl, C _3-10 heterocycloalkyl, C _6-10 aryl, C _6-10 arylalkyl, C _3-10 heteroaryl, C3-10 heteroarylalkyl, halo, oxo, -OH, -O-(C _1-4 alkyl), -O-(C _1-4 haloalkyl), -SH, -S-(C _1-4 alkyl), -S-(C _1-4 haloalkyl), -S(O)2-(C _1-4 alkyl), -S(O)2-(C _1-4 haloalkyl), -S(O)2NR ^a R ^a , -NR ^a R ^a , -NO ₂ -CN, -C(O)NR ^a R ^a , or–NR ^a C(O)R ^a . In some embodiments, R ^a can be independently selected from the group consisting of hydrogen, alkyl, haloalkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl.

[0143] In some embodiments, R ¹ can be phenyl and Q can be C _1-4 alkyl. For example, in some embodiments, R ¹ can be phenyl and Q can be methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl. In some embodiments, R ¹ can be phenyl and Q can be C _1-4 haloalkyl. For example, in some embodiments, R ¹ can be phenyl and Q can be–CF ₃ , -CF ₂ H, -CFH ₂ , -C ₂ F ₅ , -C ₂ F ₄ H, -C ₂ F ₃ H ₂ , -C ₂ F ₂ H ₃ , -C ₂ FH ₄ , or–CH(CF ₃ ) ₂ . In some embodiments, R ¹ can be phenyl and Q can be -OH. In some embodiments, R ¹ can be phenyl and Q can be -O-(C _1-4 alkyl). For example, in some embodiments, R ¹ can be phenyl and Q can be–O-methyl, -O-ethyl, -O-n-propyl, -O-isopropyl, -O-n-butyl, -O-sec-butyl, -O- isobutyl, or -O-tert-butyl. In some embodiments, R ¹ can be phenyl and Q can be C _1-4 haloalkyl. For example, in some embodiments, R ¹ can be phenyl and Q can be–O-CF ₃ ,–O- CF2H,–O-CFH2,–O-C2F5,–O-C2F4H,–O-C2F3H2,–O-C2F2H3, –O-C2FH4, or–O-CH(CF3)2.

[0144] In some embodiments, R ¹ can be phenyl and Q can be C3-6 cycloalkyl. In some embodiments, R ¹ can be phenyl and Q can be C3-10 heterocycloalkyl. In some embodiments, R ¹ can be phenyl and Q can be C _6-10 aryl. In some embodiments, R ¹ can be phenyl and Q can be C6-10 arylalkyl. In some embodiments, R ¹ can be phenyl and Q can be C _3-10 heteroaryl. In some embodiments, R ¹ can be phenyl and Q can be C _3-10 heteroarylalkyl. In some embodiments, R ¹ can be phenyl and Q can be halo. .In some embodiments, R ¹ can be phenyl and Q can be -SH. In some embodiments, R ¹ can be phenyl and Q can be -S-(C1- ₄ alkyl). In some embodiments, R ¹ can be phenyl and Q can be -S-(C _1-4 haloalkyl). In some embodiments, R ¹ can be phenyl and Q can be -S(O)2-(C _1-4 alkyl). In some embodiments, R ¹ can be phenyl and Q can be -S(O)2-(C _1-4 haloalkyl). In some embodiments, R ¹ can be phenyl and Q can be -S(O) ₂ NR ^a R ^a , -NR ^a R ^a , -C(O)NR ^a R ^a , or–NR ^a C(O)R ^a , wherein each R ^a can be independently selected from hydrogen, alkyl, haloalkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl.

[0145] In some embodiments, R ¹ can be pyridinyl and Q can be C _1-4 alkyl. For example, in some embodiments, R ¹ can be pyridinyl and Q can be methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl. In some embodiments, R ¹ can be pyridinyl and Q can be C _1-4 haloalkyl. For example, in some embodiments, R ¹ can be pyridinyl and Q can be –CF3, -CF2H, -CFH2, -C2F5, -C2F4H, -C2F3H2, -C2F2H3, -C2FH4, or –CH(CF ₃ ) ₂ . In some embodiments, R ¹ can be pyridinyl and Q can be -OH. In some embodiments, R ¹ can be pyridinyl and Q can be -O-(C _1-4 alkyl). For example, in some embodiments, R ¹ can be pyridinyl and Q can be–O-methyl, -O-ethyl, -O-n-propyl, -O- isopropyl, -O-n-butyl, -O-sec-butyl, -O-isobutyl, or -O-tert-butyl. In some embodiments, R ¹ can be pyridinyl and Q can be C _1-4 haloalkyl. For example, in some embodiments, R ¹ can be pyridinyl and Q can be–O-CF3,–O-CF2H,–O-CFH2,–O-C2F5,–O-C2F4H,–O-C2F 3H2, –O-C ₂ F ₂ H ₃ ,–O-C ₂ FH ₄ , or–O-CH(CF ₃ ) ₂ .

[0146] In some embodiments, R ¹ can be pyridinyl and Q can be C3-6 cycloalkyl. In some embodiments, R ¹ can be pyridinyl and Q can be C3-10 heterocycloalkyl. In some embodiments, R ¹ can be pyridinyl and Q can be C _6-10 aryl. In some embodiments, R ¹ can be pyridinyl and Q can be C6-10 arylalkyl. In some embodiments, R ¹ can be pyridinyl and Q can be C3-10 heteroaryl. In some embodiments, R ¹ can be pyridinyl and Q can be C3-10 heteroarylalkyl. In some embodiments, R ¹ can be pyridinyl and Q can be halo. .In some embodiments, R ¹ can be pyridinyl and Q can be -SH. In some embodiments, R ¹ can be pyridinyl and Q can be -S-(C _1-4 alkyl). In some embodiments, R ¹ can be pyridinyl and Q can be -S-(C _1-4 haloalkyl). In some embodiments, R ¹ can be pyridinyl and Q can be -S(O) ₂ -(C _1-4 alkyl). In some embodiments, R ¹ can be pyridinyl and Q can be -S(O)2-(C _1-4 haloalkyl). In some embodiments, R ¹ can be pyridinyl and Q can be -S(O)2NR ^a R ^a , -NR ^a R ^a , -C(O)NR ^a R ^a , or–NR ^a C(O)R ^a , wherein each R ^a can be independently selected from hydrogen, alkyl, haloalkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl.

[0147] In some embodiments, the compound of Formula I can be a compound of

Formula Ia having the structure including pharmaceutically acceptable salts thereof. In some embodiments, n can be 1, 2, or 3.

[0148] In some embodiments, the compound of Formula I can be a compound of

Formula Ib having the structure including pharmaceutically acceptable salts thereof. In some embodiments, m can be 0, 1, 2, 3, 4, or 5.

[0149] In some embodiments, the compound of Formula I can be a compound selected from the group consisting of:

, and pharmaceutically acceptable salts thereof.

[0150] In some embodiments, the pharmaceutically acceptable salt can be an alkaline metal salt. In some embodiments, the pharmaceutically acceptable salt can be an alkali metal salt. In some embodiments, the pharmaceutically acceptable salt can be an alkali earth metal salt. In some embodiments, the pharmaceutically acceptable salt can be an ammonium salt.

Syntheses

[0151] Compounds of Formula I, or pharmaceutically acceptable salts thereof described herein may be prepared in various ways, including those known to those skilled in the art. The routes shown and described herein are illustrative only and are not intended, nor are they to be construed, to limit the scope of the claims in any manner whatsoever. Those skilled in the art will be able to recognize modifications of the disclosed syntheses and to devise alternate routes based on the disclosures herein; all such modifications and alternate routes are within the scope of the claims. Examples of methods are described in the Examples below.

Methods of Preparation

[0152] The compounds disclosed herein may be synthesized by methods described below, or by modification of these methods. Ways of modifying the methodology include, among others, temperature, solvent, reagents etc., and will be obvious to those skilled in the art. In general, during any of the processes for preparation of the compounds disclosed herein, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry (ed. J.F.W. McOmie, Plenum Press, 1973); and Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which are both hereby incorporated herein by reference in their entirety. The protecting groups may be removed at a convenient subsequent stage using methods known from the art. Synthetic chemistry transformations useful in synthesizing applicable compounds are known in the art and include e.g. those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers, 1989, or L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons, 1995, which are both hereby incorporated herein by reference in their entirety.

[0153] Where the processes for the preparation of the compounds disclosed herein give rise to mixtures of stereoisomers, such isomers may be separated by conventional techniques such as preparative chiral chromatography. The compounds may be prepared in racemic form or individual enantiomers may be prepared by stereoselective synthesis or by resolution. The compounds may be resolved into their component enantiomers by standard techniques, such as the formation of diastereomeric pairs by salt formation with an optically active acid, such as (-)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-l-tartaric acid, followed by fractional crystallization and regeneration of the free base. The compounds may also be resolved using a chiral auxiliary by formation of diastereomeric derivatives such as esters, amides or ketals followed by chromatographic separation and removal of the chiral auxiliary. TABLE 1

Pharmaceutical Compositions

[0154] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a physiologically acceptable surface active agents, carriers, diluents, excipients, smoothing agents, suspension agents, film forming substances, and coating assistants, or a combination thereof; and a compound disclosed herein. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), which is incorporated herein by reference in its entirety. Preservatives, stabilizers, dyes, sweeteners, fragrances, flavoring agents, and the like may be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid and esters of p- hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used. In various embodiments, alcohols, esters, sulfated aliphatic alcohols, and the like may be used as surface active agents; sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium methasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like may be used as excipients; magnesium stearate, talc, hardened oil and the like may be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya may be used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of a carbohydrate such as cellulose or sugar, or methylacetate-methacrylate copolymer as a derivative of polyvinyl may be used as suspension agents; and plasticizers such as ester phthalates and the like may be used as suspension agents. [0155] The term“pharmaceutical composition,” as used herein, refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, injection, aerosol, parenteral, and topical administration. Pharmaceutical compositions can also be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

[0156] The term“carrier,” as used herein, refers to a chemical compound that facilitates the incorporation of a compound into cells or tissues. For example dimethyl sulfoxide (DMSO) is a commonly utilized carrier as it facilitates the uptake of many organic compounds into the cells or tissues of an organism.

[0157] The term“diluent,” as used herein, refers to chemical compounds diluted in water that will dissolve the compound of interest as well as stabilize the biologically active form of the compound. Salts dissolved in buffered solutions are utilized as diluents in the art. One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a compound.

[0158] The term“physiologically acceptable,” as used herein, refers to a carrier or diluent that does not abrogate the biological activity and properties of the compound.

[0159] As used herein, an“excipient” refers to an inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition. A“diluent” is a type of excipient.

[0160] For each of the compounds described herein, and for each genus or sub- genus of compounds described herein, also described are pharmaceutical compositions comprising the compound, alone or in a mixture with other compounds of the genus or sub- genus, or with alternative compounds described herein, or with one or more alternative pharmaceutically active compounds, and one or more pharmaceutically acceptable carrier, diluent, excipient or combination thereof. The pharmaceutical compositions described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or carriers, diluents, excipients or combinations thereof. Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art.

[0161] The pharmaceutical compositions disclosed herein may be manufactured in any manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes. Additionally, the active ingredients are contained in an amount effective to achieve its intended purpose. Many of the compounds used in the pharmaceutical combinations disclosed herein may be provided as salts with pharmaceutically compatible counterions.

[0162] The pharmaceutical compositions described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or suitable carriers or excipient(s). Techniques for formulation and administration of the compounds of the instant application may be found in“Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, 18th edition, 1990.

[0163] Suitable routes of administration may, for example, include oral, rectal, transmucosal, topical, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections. The compounds can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, pills, transdermal (including electrotransport) patches, and the like, for prolonged and/or timed, pulsed administration at a predetermined rate.

[0164] The pharmaceutical compositions of the present disclosure may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tabletting processes. [0165] Pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington’s Pharmaceutical Sciences, above.

[0166] Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. Physiologically compatible buffers include, but are not limited to, Hanks’s solution, Ringer’s solution, or physiological saline buffer. If desired, absorption enhancing preparations (for example, liposomes), may be utilized.

[0167] For transmucosal administration, penetrants appropriate to the barrier to be permeated may be used in the formulation.

[0168] Pharmaceutical formulations for parenteral administration, e.g., by bolus injection or continuous infusion, include aqueous solutions of the active compounds in water- soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or other organic oils such as soybean, grapefruit or almond oils, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0169] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0170] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

[0171] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0172] For administration by inhalation, the compounds for use according to the present disclosure are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0173] Further disclosed herein are various pharmaceutical compositions well known in the pharmaceutical art for uses that include intraocular, intranasal, and intraauricular delivery. Suitable penetrants for these uses are generally known in the art. Pharmaceutical compositions for intraocular delivery include aqueous ophthalmic solutions of the active compounds in water-soluble form, such as eyedrops, or in gellan gum (Shedden et al., Clin. Ther., 23(3):440-50 (2001)) or hydrogels (Mayer et al., Ophthalmologica, 210(2):101-3 (1996)); ophthalmic ointments; ophthalmic suspensions, such as microparticulates, drug-containing small polymeric particles that are suspended in a liquid carrier medium (Joshi, A., J. Ocul. Pharmacol., 10(1):29-45 (1994)), lipid-soluble formulations (Alm et al., Prog. Clin. Biol. Res., 312:447-58 (1989)), and microspheres (Mordenti, Toxicol. Sci., 52(1):101-6 (1999)); and ocular inserts. All of the above-mentioned references, are incorporated herein by reference in their entireties. Such suitable pharmaceutical formulations are most often and preferably formulated to be sterile, isotonic and buffered for stability and comfort. Pharmaceutical compositions for intranasal delivery may also include drops and sprays often prepared to simulate in many respects nasal secretions to ensure maintenance of normal ciliary action. As disclosed in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), which is incorporated herein by reference in its entirety, and well-known to those skilled in the art, suitable formulations are most often and preferably isotonic, slightly buffered to maintain a pH of 5.5 to 6.5, and most often and preferably include antimicrobial preservatives and appropriate drug stabilizers. Pharmaceutical formulations for intraauricular delivery include suspensions and ointments for topical application in the ear. Common solvents for such aural formulations include glycerin and water.

[0174] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0175] In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0176] For hydrophobic compounds, a suitable pharmaceutical carrier may be a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. A common cosolvent system used is the VPD co-solvent system, which is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co- solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of POLYSORBATE 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

[0177] Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

[0178] Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external micro-environment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. The liposome may be coated with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the desired organ. Alternatively, small hydrophobic organic molecules may be directly administered intracellularly.

[0179] Additional therapeutic or diagnostic agents may be incorporated into the pharmaceutical compositions. Alternatively or additionally, pharmaceutical compositions may be combined with other compositions that contain other therapeutic or diagnostic agents. Parenteral Pharmaceutical Composition

[0180] To prepare a parenteral pharmaceutical composition suitable for administration by injection (subcutaneous, intravenous, or the like), 0.1 mg to 100 mg of a water-soluble salt/soluble material itself/solubilized complex of a compound of a preferred embodiment is dissolved in sterile water and then mixed with 10 µof 0.9% sterile saline. The mixture is incorporated into a dosage unit form suitable for administration by injection.

Injectable Pharmaceutical Composition

[0181] To prepare an injectable formulation, 0.1 mg to 100 mg of a compound of Formula I, 2.0 mL of sodium acetate buffer solution (0.4 M), HCl (1 N) or NaOH (1 M) (q.s. to suitable pH), water (distilled, sterile) (q.s. to 20 mL) are mixed. All of the above ingredients, except water, are combined and stirred and if necessary, with slight heating if necessary. A sufficient quantity of water is then added.

Oral Pharmaceutical Composition

[0182] To prepare a pharmaceutical composition for oral delivery, 0.1 mg to 100 mg of a compound of a preferred embodiment is mixed with 750 mg of starch. The mixture is incorporated into an oral dosage unit, such as a hard gelatin capsule, or 0.1 mg to 100 mg of compound is granulated with binder solution such as starch solution along with suitable diluents such as microcrystalline cellulose or like, disintegrants such as croscaramellose sodium, dry the resultant mixture and add lubricant and compress into tablet which is suitable for oral administration.

Sublingual (Hard Lozenge) Pharmaceutical Composition

[0183] To prepare a pharmaceutical composition for buccal delivery, such as a hard lozenge, 0.1 mg to 100 mg of a compound of a preferred embodiment is mixed with 420 mg of powdered sugar/mannitol/xylitol or such sugars that provide negative heat of solution to the system, 1.6 mL of light corn syrup, 2.4 mL distilled water, and 0.42 mL mint extract or other flavorants. The mixture is blended and poured into a mold to form a lozenge suitable for buccal administration.

Fast-Disintegrating Sublingual Tablet

[0184] A fast-disintegrating sublingual tablet is prepared by mixing 48.5% by weight of a compound of a preferred embodiment, 20% by weight of microcrystalline cellulose (KG-802), 24.5% by weight of either mannitol or modified dextrose or combination that help dissolve the compressed tablet faster in the mouth, 5% by weight of low-substituted hydroxypropyl cellulose (50 ^m), and 2% by weight of magnesium stearate. Tablets are prepared by direct compression (AAPS PharmSciTech. 2006; 7(2):E41). The total weight of the compressed tablets is maintained at 150 mg. The formulation is prepared by mixing the amount of the compound of a preferred embodiment with the total quantity of microcrystalline cellulose (MCC) and mannitol/modified dextrose or combination, and two- thirds of the quantity of low-substituted hydroxypropyl cellulose (L-HPC) by using a three dimensional manual mixer (Inversina, Bioengineering AG, Switzerland) for 4.5 minutes. All of the magnesium stearate (MS) and the remaining one-third of the quantity of L-HPC are added 30 seconds before the end of mixing.

Inhalation Pharmaceutical Composition

[0185] To prepare a pharmaceutical composition for inhalation delivery, 0.1 mg to 100 mg of a compound of a preferred embodiment is mixed with 50 mg of anhydrous citric acid and 100 mL of 0.9% sodium chloride solution. The mixture is incorporated into an inhalation delivery unit, such as a nebulizer, which is suitable for inhalation administration. Nebulizer Suspension Pharmaceutical Composition

[0186] In another embodiment, a compound of a preferred embodiment (0.1 mg to 100 mg) is suspended in sterile water (100 mL); Span 85 (1 g) is added followed by addition of dextrose (5.5 g) and ascorbic acid (10 mg). Benzalkonium chloride (3 mL of a 1:750 aqueous solution) is added and the pH is adjusted to 7 with phosphate buffer. The suspension is packaged in sterile nebulizers.

Transdermal Patch Pharmaceutical Composition

[0187] To prepare a pharmaceutical composition for transdermal delivery, 0.1 mg to 100 mg of a compound of a preferred embodiment is embedded in, or deposited on, a patch with a single adhesive face. The resulting patch is then attached to the skin via the adhesive face for transdermal administration.

Topical Gel Pharmaceutical Composition

[0188] To prepare a pharmaceutical topical gel composition, 0.1 mg to 100 mg of a compound of a preferred embodiment is mixed with 1.75 g of hydroxypropyl cellulose, 10 mL of propylene glycol, 10 mL of isopropyl myristate and 100 mL of purified alcohol USP. The resulting gel mixture is then incorporated into containers, such as tubes, which are suitable for topical administration.

Ophthalmic Solution

[0189] To prepare a pharmaceutical ophthalmic solution composition, 0.1 mg to 100 mg of a compound of a preferred embodiment is mixed with 0.9 g of NaCl in 100 mL of purified water and filtered using a 0.2 micron filter. The resulting isotonic solution is then incorporated into ophthalmic delivery units, such as eye drop containers, which are suitable for ophthalmic administration. Nasal Spray Solution

[0190] To prepare a pharmaceutical nasal spray solution, 0.1 mg to 100 mg of a compound of a preferred embodiment is mixed with 30 mL of a 0.05M phosphate buffer solution (pH 4.4). The solution is placed in a nasal administrator designed to deliver 100 ^l of spray for each application.

Methods of Use

[0191] Some embodiments disclosed herein relate to a method of treating and/or ameliorating IL-6 that can include administering to a subject suffering from immunosuppression an effective amount of one or more compounds described herein (such as a compound of Formula I, or a pharmaceutically acceptable salt thereof), or a pharmaceutical composition that includes a compound described herein (such as a compound of Formula I, or a pharmaceutically acceptable salt thereof). Other embodiments disclosed herein relate to a method of treating and/or ameliorating immunosuppression that can include administering to a subject identified as suffering from immunosuppression an effective amount of one or more compounds described herein (such as a compound of Formula I, or a pharmaceutically acceptable salt thereof), or a pharmaceutically acceptable salt of any of the foregoing), or a pharmaceutical composition that includes a compound described herein such as a compound of Formula I, or a pharmaceutically acceptable salt thereof).

[0192] Some embodiments described herein relate to using one or more compounds described herein (such as a compound of Formula I, or a pharmaceutically acceptable salt thereof), in the manufacture of a medicament for ameliorating and/or treating autoimmune diseases that can include administering to a subject an effective amount of one or more compounds described herein (such as a compound of Formula I, or a pharmaceutically acceptable salt thereof)). Still other embodiments described herein relate to one or more compounds described herein (such as a compound of Formula I, or a pharmaceutically acceptable salt thereof)) that can be used for ameliorating and/or treating autoimmune diseases by administering to a subject an effective amount of one or more compounds described herein, or a pharmaceutically acceptable salt thereof.

[0193] Some embodiments disclosed herein relate to methods of ameliorating and/or treating cancer that can include contacting a cancerous cell an effective amount of one or more compounds described herein (such as a compound of Formula I, or a pharmaceutically acceptable salt thereof)), or a pharmaceutical composition that includes one or more compounds described herein (such as a compound of Formula I, or a pharmaceutically acceptable salt thereof)). In some embodiments, a compound of Formula I, or a pharmaceutically acceptable salt thereof, can act as an IL-6 antagonist, and thus, inhibit an anti-IL-6 receptor antibody.

Methods of Administration

[0194] The compounds or pharmaceutical compositions may be administered to the patient by any suitable means. Non-limiting examples of methods of administration include, among others, (a) administration though oral pathways, which administration includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (b) administration through non-oral pathways such as rectal, vaginal, intraurethral, intraocular, intranasal, or intraauricular, which administration includes administration as an aqueous suspension, an oily preparation or the like or as a drip, spray, suppository, salve, ointment or the like; (c) administration via injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, intraorbitally, intracapsularly, intraspinally, intrasternally, or the like, including infusion pump delivery; (d) administration locally such as by injection directly in the renal or cardiac area, e.g., by depot implantation; as well as (e) administration topically; as deemed appropriate by those of skill in the art for bringing the compound of the disclosure into contact with living tissue.

[0195] Pharmaceutical compositions suitable for administration include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. The therapeutically effective amount of the compounds disclosed herein required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0196] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.

[0197] In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear. The dosage may range broadly, depending upon the desired effects and the therapeutic indication. Typically, dosages may be between about 10 microgram/kg and 100 mg/kg body weight, preferably between about 100 microgram/kg and 10 mg/kg body weight. Alternatively dosages may be based and calculated upon the surface area of the patient, as understood by those of skill in the art.

[0198] The exact formulation, route of administration and dosage for the pharmaceutical compositions of the present disclosure can be chosen by the individual physician in view of the patient’s condition. (See e.g., Fingl et al. 1975, in“The Pharmacological Basis of Therapeutics”, which is hereby incorporated herein by reference in its entirety, with particular reference to Ch. 1, p. 1). Typically, the dose range of the composition administered to the patient can be from about 0.5 to 1000 mg/kg of the patient’s body weight. The dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the patient. In instances where human dosages for compounds have been established for at least some condition, the present disclosure will use those same dosages, or dosages that are between about 0.1% and 500%, more preferably between about 25% and 250% of the established human dosage. Where no human dosage is established, as will be the case for newly-discovered pharmaceutical compounds, a suitable human dosage can be inferred from ED50 or ID50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.

[0199] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0200] Although the exact dosage will be determined on a drug-by-drug basis, in most cases, some generalizations regarding the dosage can be made. The daily dosage regimen for an adult human patient may be, for example, an oral dose of between 0.1 mg and 2000 mg of each active ingredient, preferably between 1 mg and 500 mg, e.g.5 to 200 mg. In other embodiments, an intravenous, subcutaneous, or intramuscular dose of each active ingredient of between 0.01 mg and 100 mg, preferably between 0.1 mg and 60 mg, e.g. 1 to 40 mg is used. In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the free base. In some embodiments, the composition is administered 1 to 4 times per day. Alternatively the compositions of the disclosure may be administered by continuous intravenous infusion, preferably at a dose of each active ingredient up to 1000 mg per day. As will be understood by those of skill in the art, in certain situations it may be necessary to administer the compounds disclosed herein in amounts that exceed, or even far exceed, the above-stated, preferred dosage range in order to effectively and aggressively treat particularly aggressive diseases or infections. In some embodiments, the compounds will be administered for a period of continuous therapy, for example for a week or more, or for months or years. [0201] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

[0202] Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.

[0203] In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0204] The amount of composition administered may be dependent on the subject being treated, on the subject’s weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

[0205] Compounds disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular compound, or of a subset of the compounds, sharing certain chemical moieties, may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of particular compounds in an animal model, such as mice, rats, rabbits, or monkeys, may be determined using known methods. The efficacy of a particular compound may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. Recognized in vitro models exist for nearly every class of condition, including but not limited to cancer, cardiovascular disease, and various immune dysfunction. Similarly, acceptable animal models may be used to establish efficacy of chemicals to treat such conditions. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, and route of administration, and regime. Of course, human clinical trials can also be used to determine the efficacy of a compound in humans. [0206] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions comprising a compound of the disclosure formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Administration and Pharmaceutical Compositions

[0207] The compounds are administered at a therapeutically effective dosage. While human dosage levels have yet to be optimized for the compounds described herein, generally, a daily dose may be from about 0.25 mg/kg to about 120 mg/kg or more of body weight, from about 0.5 mg/kg or less to about 70 mg/kg, from about 1.0 mg/kg to about 50 mg/kg of body weight, or from about 1.5 mg/kg to about 10 mg/kg of body weight. Thus, for administration to a 70 kg person, the dosage range would be from about 17 mg per day to about 8000 mg per day, from about 35 mg per day or less to about 7000 mg per day or more, from about 70 mg per day to about 6000 mg per day, from about 100 mg per day to about 5000 mg per day, or from about 200 mg to about 3000 mg per day. The amount of active compound administered will, of course, be dependent on the subject and disease state being treated, the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician.

[0208] Administration of the compounds disclosed herein or the pharmaceutically acceptable salts thereof can be via any of the accepted modes of administration for agents that serve similar utilities including, but not limited to, orally, subcutaneously, intravenously, intranasally, topically, transdermally, intraperitoneally, intramuscularly, intrapulmonarilly, vaginally, rectally, or intraocularly. Oral and parenteral administrations are customary in treating the indications that are the subject of the preferred embodiments.

[0209] The compounds useful as described above can be formulated into pharmaceutical compositions for use in treatment of these conditions. Standard pharmaceutical formulation techniques are used, such as those disclosed in Remington's The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005), incorporated by reference in its entirety. Accordingly, some embodiments include pharmaceutical compositions comprising: (a) a safe and therapeutically effective amount of a compound described herein (including enantiomers, diastereoisomers, tautomers, polymorphs, and solvates thereof), or pharmaceutically acceptable salts thereof; and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof.

[0210] In addition to the selected compound useful as described above, come embodiments include compositions containing a pharmaceutically-acceptable carrier. The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. In addition, various adjuvants such as are commonly used in the art may be included. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman’s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety.

[0211] Some examples of substances, which can serve as pharmaceutically- acceptable carriers or components thereof, are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions.

[0212] The choice of a pharmaceutically-acceptable carrier to be used in conjunction with the subject compound is basically determined by the way the compound is to be administered.

[0213] The compositions described herein are preferably provided in unit dosage form. As used herein, a "unit dosage form" is a composition containing an amount of a compound that is suitable for administration to an animal, preferably mammal subject, in a single dose, according to good medical practice. The preparation of a single or unit dosage form however, does not imply that the dosage form is administered once per day or once per course of therapy. Such dosage forms are contemplated to be administered once, twice, thrice or more per day and may be administered as infusion over a period of time (e.g., from about 30 minutes to about 2-6 hours), or administered as a continuous infusion, and may be given more than once during a course of therapy, though a single administration is not specifically excluded. The skilled artisan will recognize that the formulation does not specifically contemplate the entire course of therapy and such decisions are left for those skilled in the art of treatment rather than formulation.

[0214] The compositions useful as described above may be in any of a variety of suitable forms for a variety of routes for administration, for example, for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration. The skilled artisan will appreciate that oral and nasal compositions comprise compositions that are administered by inhalation, and made using available methodologies. Depending upon the particular route of administration desired, a variety of pharmaceutically-acceptable carriers well-known in the art may be used. Pharmaceutically-acceptable carriers include, for example, solid or liquid fillers, diluents, hydrotropies, surface-active agents, and encapsulating substances. Optional pharmaceutically-active materials may be included, which do not substantially interfere with the inhibitory activity of the compound. The amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound. Techniques and compositions for making dosage forms useful in the methods described herein are described in the following references, all incorporated by reference herein: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).

[0215] Various oral dosage forms can be used, including such solid forms as tablets, capsules, granules and bulk powders. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow- inducing agents, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.

[0216] The pharmaceutically-acceptable carrier suitable for the preparation of unit dosage forms for peroral administration is well-known in the art. Tablets typically comprise conventional pharmaceutically-compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. Glidants such as silicon dioxide can be used to improve flow characteristics of the powder mixture. Coloring agents, such as the FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically comprise one or more solid diluents disclosed above. The selection of carrier components depends on secondary considerations like taste, cost, and shelf stability, which are not critical, and can be readily made by a person skilled in the art.

[0217] Peroral compositions also include liquid solutions, emulsions, suspensions, and the like. The pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art. Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, AVICEL RC-591, tragacanth and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate. Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.

[0218] Such compositions may also be coated by conventional methods, typically with pH or time-dependent coatings, such that the subject compound is released in the gastrointestinal tract in the vicinity of the desired topical application, or at various times to extend the desired action. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, Eudragit coatings, waxes and shellac.

[0219] Compositions described herein may optionally include other drug actives.

[0220] Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included.

[0221] A liquid composition, which is formulated for topical ophthalmic use, is formulated such that it can be administered topically to the eye. The comfort should be maximized as much as possible, although sometimes formulation considerations (e.g. drug stability) may necessitate less than optimal comfort. In the case that comfort cannot be maximized, the liquid should be formulated such that the liquid is tolerable to the patient for topical ophthalmic use. Additionally, an ophthalmically acceptable liquid should either be packaged for single use, or contain a preservative to prevent contamination over multiple uses.

[0222] For ophthalmic application, solutions or medicaments are often prepared using a physiological saline solution as a major vehicle. Ophthalmic solutions should preferably be maintained at a comfortable pH with an appropriate buffer system. The formulations may also contain conventional, pharmaceutically acceptable preservatives, stabilizers and surfactants.

[0223] Preservatives that may be used in the pharmaceutical compositions disclosed herein include, but are not limited to, benzalkonium chloride, PHMB, chlorobutanol, thimerosal, phenylmercuric, acetate and phenylmercuric nitrate. A useful surfactant is, for example, Tween 80. Likewise, various useful vehicles may be used in the ophthalmic preparations disclosed herein. These vehicles include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose and purified water.

[0224] Tonicity adjustors may be added as needed or convenient. They include, but are not limited to, salts, particularly sodium chloride, potassium chloride, mannitol and glycerin, or any other suitable ophthalmically acceptable tonicity adjustor.

[0225] Various buffers and means for adjusting pH may be used so long as the resulting preparation is ophthalmically acceptable. For many compositions, the pH will be between 4 and 9. Accordingly, buffers include acetate buffers, citrate buffers, phosphate buffers and borate buffers. Acids or bases may be used to adjust the pH of these formulations as needed.

[0226] In a similar vein, an ophthalmically acceptable antioxidant includes, but is not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.

[0227] Other excipient components, which may be included in the ophthalmic preparations, are chelating agents. A useful chelating agent is edetate disodium, although other chelating agents may also be used in place or in conjunction with it.

[0228] For topical use, creams, ointments, gels, solutions or suspensions, etc., containing the compound disclosed herein are employed. Topical formulations may generally be comprised of a pharmaceutical carrier, co-solvent, emulsifier, penetration enhancer, preservative system, and emollient.

[0229] For intravenous administration, the compounds and compositions described herein may be dissolved or dispersed in a pharmaceutically acceptable diluent, such as a saline or dextrose solution. Suitable excipients may be included to achieve the desired pH, including but not limited to NaOH, sodium carbonate, sodium acetate, HCl, and citric acid. In various embodiments, the pH of the final composition ranges from 2 to 8, or preferably from 4 to 7. Antioxidant excipients may include sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA. Other non-limiting examples of suitable excipients found in the final intravenous composition may include sodium or potassium phosphates, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol, and dextran. Further acceptable excipients are described in Powell, et al., Compendium of Excipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998, 52 238-311 and Nema et al., Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions, PDA J Pharm Sci and Tech 2011, 65 287-332, both of which are incorporated herein by reference in their entirety. Antimicrobial agents may also be included to achieve a bacteriostatic or fungistatic solution, including but not limited to phenylmercuric nitrate, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.

[0230] The compositions for intravenous administration may be provided to caregivers in the form of one more solids that are reconstituted with a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration. In other embodiments, the compositions are provided in solution ready to administer parenterally. In still other embodiments, the compositions are provided in a solution that is further diluted prior to administration. In embodiments that include administering a combination of a compound described herein and another agent, the combination may be provided to caregivers as a mixture, or the caregivers may mix the two agents prior to administration, or the two agents may be administered separately.

[0231] The actual dose of the active compounds described herein depends on the specific compound, and on the condition to be treated; the selection of the appropriate dose is well within the knowledge of the skilled artisan.

Second (or Other Additional) Agents

[0232] In some embodiments, the second therapeutic agent is anti-inflammatory agent. In some embodiments, the second therapeutic agent is a non-steroidal anti- inflammatory agent. In some embodiments, the second therapeutic agent is anti-cancer agent. [0233] In some embodiments, the second therapeutic agent is selected from aspirin; diflunisal; salsalate; acetaminophen; ibuprofen; dexibuprofen; naproxen; fenoprofen; ketoprofen; dexketoprofen; flurbiprofen; oxaprozin; loxoprofen; indomethacin; tolmetin; sulindac; etodolac; ketorolac; diclofenac; aceclofenac; nabumetone; enolic acid; piroxicam; meloxicam; tenoxicam; droxicam; lornoxicam; isoxicam; mefenamic acid; meclofenamic acid; flufenamic acid; tolfenamic acid; sulfonanilides; clonixin; licofelone; dexamethasone; and prednisone.

[0234] In some embodiments, the second therapeutic agent is selected from mechlorethamine; cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N- nitroso-N-methylurea (MNU); carmustine (BCNU); lomustine (CCNU); semustine (MeCCNU); fotemustine; streptozotocin; dacarbazine; mitozolomide; temozolomide; thiotepa; mytomycin; diaziquone (AZQ); cisplatin; carboplatin; and oxaliplatin.

[0235] In some embodiments, the second therapeutic agent is selected from vincristine; vinblastine; vinorelbine; vindesine; vinflunine; paclitaxel; docetaxel; etoposide; teniposide; tofacitinib; ixabepilone; irinotecan; topotecan; camptothecin; doxorubicin; mitoxantrone; and teniposide.

[0236] In some embodiments, the second therapeutic agent is selected from actinomycin; bleomycin; plicamycin; mitomycin; daunorubicin; epirubicin; idarubicin; pirarubicin; aclarubicin; mitoxantrone; cyclophosphamide; methotrexate; 5-fluorouracil; prednisolone; folinic acid; methotrexate; melphalan; capecitabine; mechlorethamine; uramustine; melphalan; chlorambucil; ifosfamide; bendamustine; 6-mercaptopurine; and procarbazine.

[0237] In some embodiments, the second therapeutic agent is selected from cladribine; pemetrexed; fludarabine; gemcitabine; hydroxyurea; nelarabine; cladribine; clofarabine; ytarabine; decitabine; cytarabine; cytarabine liposomal; pralatrexate; floxuridine; fludarabine; colchicine; thioguanine; cabazitaxel; larotaxel; ortataxel; tesetaxel; aminopterin; pemetrexed; pralatrexate; raltitrexed; pemetrexed; carmofur; and floxuridine.

[0238] In some embodiments, the second therapeutic agent is selected from azacitidine; decitabine; hydroxycarbamide; topotecan; irinotecan; belotecan; teniposide; aclarubicin; epirubicin; idarubicin; amrubicin; pirarubicin; valrubicin; zorubicin; mitoxantrone; pixantrone; mechlorethamine; chlorambucil; prednimustine; uramustine; estramustine; carmustine; lomustine; fotemustine; nimustine; ranimustine; carboquone; thioTEPA; triaziquone; and triethylenemelamine.

[0239] In some embodiments, the second therapeutic agent is selected from nedaplatin; satraplatin; procarbazine; dacarbazine; temozolomide; altretamine; mitobronitol; pipobroman; actinomycin; bleomycin; plicamycin; aminolevulinic acid; methyl aminolevulinate; efaproxiral; talaporfin; temoporfin; verteporfin; alvocidib; seliciclib; palbociclib; bortezomib; carfilzomib; anagrelide; masoprocol; olaparib; belinostat; panobinostat; romidepsin; vorinosta; idelalisib; atrasentan; bexarotene; testolactone; amsacrine; trabectedin; alitretinoin; tretinoin; demecolcine; elsamitrucin; etoglucid; lonidamine; lucanthone; mitoguazone; mitotane; oblimersen; omacetaxine mepesuccinate; and eribulin.

[0240] In some embodiments, the second therapeutic agent is selected from azathioprine; Mycophenolic acid; leflunomide; teriflunomide; tacrolimus; cyclosporin; pimecrolimus; abetimus; gusperimus; lenalidomide; pomalidomide; thalidomide; anakinra; sirolimus; everolimus; ridaforolimus; temsirolimus; umirolimus; zotarolimus; eculizumab; adalimumab; afelimomab; certolizumab pegol; golimumab; infliximab; nerelimomab; mepolizumab; omalizumab; faralimomab; elsilimomab; lebrikizumab; ustekinumab; etanercept; otelixizumab; teplizumab; visilizumab; clenoliximab; keliximab; zanolimumab; efalizumab; erlizumab; obinutuzumab; rituximab; and ocrelizumab.

[0241] In some embodiments, the second therapeutic agent is selected from pascolizumab; gomiliximab; lumiliximab; teneliximab; toralizumab; aselizumab; galiximab; gavilimomab; ruplizumab; belimumab; blisibimod; ipilimumab; tremelimumab; bertilimumab; lerdelimumab; metelimumab; natalizumab; tocilizumab; odulimomab; basiliximab; daclizumab; inolimomab; zolimoma; atorolimumab; cedelizumab; fontolizumab; maslimomab; morolimumab; pexelizumab; reslizumab; rovelizumab; siplizumab; talizumab; telimomab; vapaliximab; vepalimomab; abatacept; belatacept; pegsunercept; aflibercept; alefacept; and rilonacept. EXAMPLES

General Procedures

[0242] Materials used in preparing the nifuroxazide derivatives described herein may be made by known methods or are commercially available. In these reactions, it is also possible to make use of variants which are themselves known to those of ordinary skill in this art, but are not mentioned in greater detail. The skilled artisan given the literature and this disclosure is well equipped to prepare any of the compounds.

[0243] It is recognized that the skilled artisan in the art of organic chemistry can readily carry out manipulations without further direction, that is, it is well within the scope and practice of the skilled artisan to carry out these manipulations. These include reduction of carbonyl compounds to their corresponding alcohols, oxidations, acylations, aromatic substitutions, both electrophilic and nucleophilic, etherifications, esterification and saponification and the like. These manipulations are discussed in standard texts such as March’s Advanced Organic Chemistry (Wiley), Carey and Sundberg, Advanced Organic Chemistry (incorporated herein by reference in their entirety) and the like.

[0244] The skilled artisan will readily appreciate that certain reactions are best carried out when other functionality is masked or protected in the molecule, thus avoiding any undesirable side reactions and/or increasing the yield of the reaction. Often the skilled artisan utilizes protecting groups to accomplish such increased yields or to avoid the undesired reactions. These reactions are found in the literature and are also well within the scope of the skilled artisan. Examples of many of these manipulations can be found for example in T. Greene and P. Wuts Protecting Groups in Organic Synthesis, 4th Ed., John Wiley & Sons (2007), incorporated herein by reference in its entirety.

[0245] The following example schemes are provided for the guidance of the reader, and represent preferred methods for making the compounds exemplified herein. These methods are not limiting, and it will be apparent that other routes may be employed to prepare these compounds. Such methods specifically include solid phase based chemistries, including combinatorial chemistry. The skilled artisan is thoroughly equipped to prepare these compounds by those methods given the literature and this disclosure. The compound numberings used in the synthetic schemes depicted below are meant for those specific schemes only, and should not be construed as or confused with same numberings in other sections of the application.

[0246] Trademarks used herein are examples only and reflect illustrative materials used at the time of the disclosure. The skilled artisan will recognize that variations in lot, manufacturing processes, and the like, are expected. Hence the examples, and the trademarks used in them are non-limiting, and they are not intended to be limiting, but are merely an illustration of how a skilled artisan may choose to perform one or more of the embodiments of the disclosure.

[0247] The following abbreviations have the indicated meanings:

AcOH = acetic acid

BSA = bovine serum albumin

DMSO = dimethyl sulfoxide

EtOAc = ethyl acetate

HCl = hydrochloric acid

MeOH = methanol

Na ₂ SO ₄ = sodium sulfate

NaHCO3 = sodium bicarbonate

NMR = nuclear magnetic resonance

PBS = phosphate buffered saline [0248] The following example schemes are provided for the guidance of the reader, and collectively represent an example method for making the compounds provided herein. Furthermore, other methods for preparing compounds described herein will be readily apparent to the person of ordinary skill in the art in light of the following reaction schemes and examples. Unless otherwise indicated, all variables are as defined above. EXAMPLE 1

(Z)-3-(4-HYDROXYPHENYL)-5-((5-NITROFURAN-2- YL)METHYLENE)THIAZOLIDINE-2,4-DIONE (1)

Synthesis of triethylamine (4-hydroxyphenyl)-carbamodithioate (Int-1)

[0249] To a solution of 4-aminophenol (2.5 g, 22.5 mmol) in carbon disulfide (2.1 g, 27.5 mmol) at 0 ^o C was added triethylamine (26 mL). The reaction mixture was allowed to warm to room temperature and stirred overnight. This reaction was performed three times and the three reaction mixtures were combined and concentrated to remove most of the solvent. The remaining suspension was filtered and the solid was washed with n-hexane to yield 19.5 g, (99%) of triethylamine (4-hydroxyphenyl)-carbamodithioate (Int-1) as a brown solid. NMR (400 MHz, DMSO-d6) ^ 9.83 (s, 1H), 9.00 (s, 1H), 7.52– 7.44 (m, 2H), 6.60 – 6.55 (m, 2H), 3.06– 2.95 (m, 6H), 1.15 (t, J = 7.2 Hz, 9H).

Synthesis of 3-(4-Hydroxyphenyl)-2-thioxothiazolidin-4-one (Int-3)

[0250] To a suspension of triethylamine (4-hydroxyphenyl)carbamodithioate (Int- 1) (6.0 g, 20.9 mmol) in H2O (40 mL) at 0 ^o C was added dropwise methyl 2-bromoacetate (3.2 g, 20.9 mmol). The reaction was stirred at 0 ^o C for 1 hour. A suspension formed and the solid methyl 2-(((4-hydroxyphenyl)carbamothioyl)thio)acetate (Int-2) was separated via filtration and used without further purification. [0251] The solid methyl 2-(((4-hydroxyphenyl)carbamothioyl)thio)acetate (Int-2) was suspended in H2O (50 mL) at room temperature and the mixture was heated to 100 ^o C for 3 hours. The mixture was allowed to cool to room temperature. The solid was separated via filtration to yield 4.1 g (87%) of 3-(4-hydroxyphenyl)-2-thioxothiazolidin-4-one (Int-3) as a brown solid. MS (ESI, pos. ion) m/z: 226.0 (M+1). ¹ H NMR (400 MHz, DMSO-d6) ^ 9.83 (s, 2H), 7.05– 6.99 (m, 2H), 6.88– 6.82 (m, 2H), 4.35 (s, 2H).

Synthesis of 3-(4-hydroxyphenyl)-thiazolidine-2,4-dione (Int-4)

[0252] To a mixture of 3-(4-hydroxyphenyl)-2-thioxothiazolidin-4-one (Int-3) (2.0 g, 8.9 mmol) and Na2WO4 (0.3 g, 0.9 mmol) in EtOAc (80 mL) at 0 ^o C was added dropwise H2O2 (50 mL). Additional EtOAc (20 mL) and H2O2 (20 mL) were added at 0 ^o C. The reaction mixture was slowly warmed to room temperature and stirred at room temperature for 5 hours. The organic layer was separated, washed with brine and saturated sodium sulfite, dried over Na2SO4, filtered and concentrated. The residue was purified via flash column chromatography (eluent: petroleum ether:EtOAc = 2:3) to yield 1.1 g (59%) of 3-(4-hydroxyphenyl)-thiazolidine-2,4-dione (Int-4) as gray solid. MS (ESI, pos. ion) m/z: 210.0 (M+1). ¹ H NMR (400 MHz, DMSO-d6) ^ 9.82 (s, 1H), 7.07 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 4.27 (s, 2H).

Synthesis of (Z)-3-(4-hydroxyphenyl)-5-[(5-nitrofuran-2-yl)-methylene]-th iazolidine-2,4- dione (1)

[0253] To a solution of 3-(4-hydroxyphenyl)-thiazolidine-2,4-dione (200.0 mg, 0.96 mmol) and beta-alanine (171.1 mg, 1.92 mmol) in AcOH (6 mL) was added 5- nitrofuran-2-carbaldehyde (148.4 mg, 1.1 mmol). The reaction mixture was stirred at 100 ^o C for 1 hour. Additional 5-nitrofuran-2-carbaldehyde (148.4 mg, 1.1 mmol) was added and the reaction was stirred at 100 ^o C for another 6 hours. The reaction was allowed to cool to room temperature, concentrated and purified via recrystallization (acetonitrile) to yield 35 mg (11%) of (Z)-3-(4-hydroxyphenyl)-5-[(5-nitrofuran-2-yl)-methylene]-th iazolidine-2,4-dione (1) as a yellow solid. MS (ESI, pos. ion) m/z: 333.1 (M+1). ¹ H NMR (400 MHz, DMSO-d6) ^ 9.88 (s, 1H), 7.89 (s, 1H), 7.86 (d, J = 4.0 Hz, 0H), 7.39 (d, J = 4.0 Hz, 1H), 7.25– 7.18 (m, 2H), 6.91– 6.84 (m, 2H). EXAMPLE 2

PHOSPHORYLATED-STAT3 (pSTAT3) PHARMACODYNAMIC ASSAY [0254] Engagement of interleukin-6 (IL-6) to its receptor (IL6R) and co-receptor (IL6ST) lead to activation of Signal Transducer and Activator of Transcription-3 (STAT3) via Janus Activiated Kinase (JAK)-dependent and independent mechanisms, which result in phosphorylation of tyrosine 705 (Y705). Disruption of IL-6 signaling lead to reductions in nuclear pSTAT3, and pharmacologic reductions in nuclear pSTAT3 levels, in tumor cells or immune cells, represent a pharmacodynamic (PD) biomarker associated with desired drug activity.

[0255] The pSTAT3 Pharmacodynamic Assay was performed using an ImageXpress Micro Confocal High-Content Imaging System in a 96-well format (Costar™ 96-Well Black Clear-Bottom Plates - Tissue culture treated; Cat # 07-200-588). Adjustable 1 mL, 5 mL, 10 mL, 25 mL pipettes were used for reagent preparation and Pipetmen (Precision) 50 µL and 20-200 µL or equivalent with sterile tips (e.g., Eppendorf) were used to dispense media for the assay. Cells were counted using a TC10 cell counter or hemocytometer.

[0256] Tumor cells were counted using a hemocyometer or cell counter and plated at 8,000 cells/well in complete medium (RPMI-1640 (ThermoFisher #11835030; pH 6.8); 10% FBS (HyClone # SH30910.03); 100U/mL Pen/Strep (ThermoFisher #15140122) in a 96-well plate. Caution was taken to avoid plating in the outer wells to prevent any cell stress or dehydration. After 48 h in culture (based on cell health and number), the culture medium was removed and RPMI-1640 (ThermoFisher #11835030; pH 6.8) without phenol red to wash the cells. The cells were diluted in RPMI-1640 without phenol red and incubated at 37 ºC for 1 hour and 50 ng/mL recombinant interleukin-6 (IL-6) for 0.5 hours.

[0257] After the drug treatment, the medium was removed and replaced with 100 µL/well of a 7.4% formaldehyde fixative solution and incubated at room temperature for 10 minutes. The fixative formaldehyde solution was removed and the cells were washed twice with PBS followed by permeabilization using 100% ice-cold methanol (100 µL/well) at 4 ºC for 30 min. The permeablized cells were washed twice with PBS and blocked with 5% BSA, 0.1% Tween-20/PBS (100 µL/well) for 30 min at room temperature. After blocking, the cells were washed twice with 0.1% Tween-20/PBS. The primary antibody was diluted at 1:100 v/v in 1% BSA, 0.1% Tween-20/PBS and incubated over wells (50 µL/well) overnight at 4 ºC.

[0258] The primary antibody was washed off and the cells were washed twice with 0.1% Tween-20/PBS (100 µL/well). The secondary antibody at 1:500 (v/v) in PBS/0/.1% Tween-20) and incubate at room temperature for 1 hour. The cells were then washed twice with 100 µL/well 0.1% Tween-20/PBS. After washing, 100 µL of 4',6- diamidino-2-phenylindole (DAPI; Sigma Cat # D8417, diluted at 1:1000 v/v) was added to the wells and incubated for 10 minutes. DAPI was washed off twice using 100 µL PBS and the plates using the High Content Imager. The results are presented in Table 2 below. TABLE 2: pSTAT3 results

* As a percentage of control

# The effective concentration (EC) that produced half (EC50) of the maximum achieved pSTAT3 effect at 10 µM doses (cited above) were: EC50 < 500 nM (MCF-7); < 500 nM (BT- 474); <1.00 µM (SK-BR-3) EXAMPLE 3

HUMANIZED 3D-TUMOR GROWTH ASSAY (3D-TGA) Materials

[0259] The following materials are used for the Humanized 3D-Tumor Growth Assay: DPBS (GE Healthcare SH3002802); Trypsin-EDTA (Cellgro 25051.CL); MRT 3D- TGA Matrix Medium Plus (custom); MRT custom Basement Membrane Extract (BME); 96- well plates, black-walled, clear-bottomed, not tissue culture treated, and sterile (BrandTech, 781671); 5α-Androstan-17β-ol-3-one / Di-hydrotestosterone (DHT) (Sigma, A8380); β- Estradiol (E2) (Sigma, E2758); Progesterone (Sigma P8783); L-glutamine (Gibco, 25030081); Alamar blue (Invitrogen, DAL1100); Plate warmer (VWR 460-3253 & 460- 3238); Formaldehyde (Sigma-Aldrich, F8875); Sulfo-Cy5-Azide (Lumiprobe, A3330): 4 mM in H ₂ O; CuSO ₄ .5H ₂ O (Sigma, 12849): 200 mM in H ₂ O; Ascorbic Acid (Sigma, A4544): 200mg/mL in H2O (FRESH); Triton-X (ACROS Organics, 327371000); DAPI (Invitrogen, D1306): 40 mM in DMF; and EdU (Lumiprobe, 10540)

Procedure

[0260] Note: Before performing any tissue culture work, carefully plan template to calculate quantity of BME needed. BME should be thawed at 4 ^o C overnight, but it can be thawed more quickly by placing it on ice for approximately three to four hours.

[0261] Generate a single cell suspension of tumor cells by following a standard subculturing protocol for adherent cells.

[0262] Aspirate media from flask or culture dish.

[0263] Wash with enough sterile DPBS to cover the bottom of the vessel, then aspirate.

[0264] Add enough Trypsin-EDTA to cover the bottom of the vessel, then place back in tissue culture incubator at 37 ^o C for 5 to 10 minutes, or until cells have detached from the culture vessel.

[0265] Verify cells have detached from surface by observation using an inverted phase contrast microscope.

[0266] Once all cells have detached from the vessel, add a at least one volume of complete media with serum (standard 2D tissue culture media) to neutralize the trypsin.

[0267] Transfer cells to an appropriately sized conical tube for centrifugation at 200g for 5 minutes

[0268] While pelleting cells, set plate warmer to 37 ^o C and place clear bottom, black walled plate on warmer.

[0269] Add 200 µl of sterile PBS to all outer wells. [0270] Aspirate supernatant from cell pellet and resuspend in an appropriate amount of media.

[0271] Assess the viability of the cell suspension by haemocytometer or automated cell counter.

[0272] Transfer desired quantity of cells to a sterile 1.5mL Eppendorf or 15mL universal, as appropriate.

[0273] Pellet cells (5min, 200g).

[0274] Carefully aspirate all but approximately 10 µL medium from pellet taking extreme care to avoid the pellet.

[0275] NOTE: Do this by hand, do NOT use aspirator.

[0276] Suspend all cell pellets in 50µL/well in MRT 3D-TGA Matrix Medium Plus kept on ice or at 4 ^o C.

[0277] Note: Prepare hood with all equipment needed, from this point work quickly and accurately. If working with a large number of wells, use repeater pipette.

[0278] Add an equal volume of working stock BME and carefully pipette up and down to mix. Avoid introducing bubbles.

[0279] Add 100µL cell suspension/well into wells (with plate on 37 ^o C plate warmer) taking caution to avoid bubbles.

[0280] NOTE: make sure cells are suspended thoroughly and use positive displacement pipetting to avoid bubbles.

[0281] Gently tap plate or use a syringe needle to pop any bubbles.

[0282] Place 96-well plate in tri-gas incubator (5% CO2, 6% O2 and 90% N2) at 37 ^o C.

Day 1

[0283] Apply drug(s) of interest 24 hours after seeding.

[0284] Drugs should be diluted into Matrix Media Plus, supplemented with hormone cocktail consisting of E2 (50 pg/mL final in well), DHT (250 pg/mL final in well), and progesterone (1 ng/mL final in well). Add L-glutamine to 2 mM final concentration.

[0285] Working stock of drug dilution should be at 3x the desired final concentration. [0286] All required volumes should be calculated based on adding 50 µL/well, giving a final volume of 150 µL/well post application, with enough overage to accommodate 5% for pipetting error.

Day 2

[0287] Apply EdU 48 hours after seeding.

[0288] Add an additional 50 µL/well of matrix media plus, supplemented with hormones and 40 µM EdU to each well, giving a final concentration of 10 µM EdU in each well, and a final volume of 200 µL in each well.

[0289] Note: wells not receiving EdU treatment will need 50 µL of matrix media plus added to them as well.

Day 4

[0290] Perform alalmarBlue assay

[0291] Calculate volume of alamarBlue required based on 10µL/well.

[0292] Dilute alamarBlue 1:1 in matrix media plus and add 20µL/well to all wells.

[0293] Incubate at 37 ^o C in tri-gas incubator for exactly 180 minutes.

[0294] Immediately read on the Molecular Devices Flexstation III (or similar platereader) using alamarBlue Ex:Em settings:

[0295] Excitation: 560 Emission: 588 Cutoff: 570

[0296] Bottom read, Endpoint read.

Process for Cell Proliferation‘ClickIT chemistry’ and EdU detection

[0297] Determine the volume of fix solution based on 100 µL per well.

[0298] Prepare fix solution by mixing 37% formaldehyde solution 1:10 with 1X PBS (prepare fresh every day).

[0299] Remove medium from wells to be fixed, ~100µL, being careful to avoid disturbing BME.

[0300] Gently wash 1x w/ 100 µl of PBS.

[0301] Add 100 µl of fixation, incubate RT in dark 15-20 minutes.

[0302] Gently wash 2x with PBS:

[0303] First wash is quick, on and off with 100 µl of PBS [0304] Second wash is longer, 5-10 minutes RT in dark with 200 µl PBS

[0305] NOTE: Plate can be stored at this point by adding 100 µl of PBS to each well and placing in the dark at 4 ^o C. PBS used for storage must be removed before beginning permeabilization process.

[0306] Determine volume of permeabilization solution based on 100 µl per well.

[0307] Prepare 0.1% Triton X-100 in PBS permeabilization solution and filter through a 1 µm syringe filter.

[0308] Add 100 µl of permeabilization buffer to each well and incubate 15-20 minutes at RT in dark.

[0309] Gently wash 2x with PBS as above.

[0310] NOTE: Keep the last wash on and the plate in the dark until the ClickIT chemistry reagent cocktail is ready.

[0311] Determine total volume of ClickIT chemistry reaction cocktail based on 50 µl per well.

[0312] Prepare cocktail by combining, in this order:

[0313] PBS– 888 µL

[0314] CuSO4– 10 µL

[0315] Azide-dye– 2 µL

[0316] Ascorbate– 100 µL

[0317] NOTE: 1mL of cocktail is good for 20 wells. Cocktail is good for 24 hours stored at 4 ^o C in the dark.

[0318] Remove PBS, then add 50 µL of cocktail to each well and incubate at RT in dark for 30-60 minutes.

[0319] During ClickIT reaction incubation, determine volume of nuclear staining solution required (50 µL per well), and prepare 1x DAPI solution by diluting DAPI 1:1000 in PBS.

[0320] Gently wash 2x with PBS as above.

[0321] Add 50 µL of nuclear staining solution to each well and incubate at RT in the dark for 15 minutes.

[0322] Gently wash 2x with PBS as above [0323] Add 50 µl of PBS before analysis and keep in dark for a minimum of 30 minutes before analysis.

[0324] The results of the 3D-TGA are provided in Table 4 below. TABLE 3: 3D-TGA results

EXAMPLE 4

STANDARD 2D TISSUE CULTURE ASSAY Materials

[0325] The following materials were used in the Standard 2DPBS (GE Healthcare SH30028.02): Trypsin-EDTA (Cellgro 25051.CL); RPMI-1640 (Gibco, 11835030); Fetal bovine serum (GE Healthcare, SH30910.03); Gluta-max (Gibco, 35050061); Penicillin- Streptomycin (Gibco, 15140122); 96-well plates, black-walled, clear-bottomed, tissue culture treated, and sterile: (Corning, 3904); Alamar blue (Invitrogen DAL1100); Formaldehyde (Sigma-Aldrich, F8875); Sulfo-Cy5-Azide (Lumiprobe, A3330): 4 mM in H2O; CuSO4.5H2O (Sigma, 12849): 200 mM in H2O; and Ascorbic Acid (Sigma, A4544): 200mg/mL (FRESH) Procedure

Day 0

[0326] Generate a single cell suspension of tumor cells by following a standard subculturing protocol for adherent cells.

[0327] Aspirate media from flask or culture dish.

[0328] Wash with enough sterile DPBS to cover the bottom of the vessel, then aspirate.

[0329] Add enough Trypsin-EDTA to cover the bottom of the vessel, then place back in tissue culture incubator at 37 ^o C for 5 to 10 minutes, or until cells have detached from the culture vessel.

[0330] Verify cells have detached from surface by observation using an inverted phase contrast microscope.

[0331] Once all cells have detached from the vessel, add a at least two volumes of complete media with serum to neutralize the trypsin.

[0332] Transfer cells to an appropriately sized conical tube for centrifugation at 200g for 5 minutes.

[0333] While pelleting cells, add 200µl of PBS to outer wells of a clear bottom, black walled plate.

[0334] Aspirate supernatant from cell pellet and suspend in an appropriate amount of media.

[0335] Assess the viability of the cell suspension by haemocytometer or automatic cell counter.

[0336] Count cells and calculate number of tumor cells add per group.

[0337] Transfer desired quantity of cells to a sterile 1.5mL Eppendorf or 15mL universal, as appropriate.

[0338] Pellet cells (5min, 200g).

[0339] Carefully aspirate all but ~10µL medium from pellet taking extreme care to avoid the pellet. NOTE: Do this by hand, do NOT use aspirator. [0340] Suspend all cell pellets in a volume of media equal to 100µl /well in RPMI-1640 (phenol red free, with 10% FBS, 100U/mL penicillin-streptomycin and 2mM Glutamax supplementation). Transition suspended cells to a multichannel pipette reservoir.

[0341] Note: Prepare hood with all equipment needed, from this point work quickly and accurately. If working with a large number of wells use repeater pipette.

[0342] Add 100µl cell suspension/well into desired wells.

[0343] NOTE: make sure cells are mixed thoroughly.

[0344] Place 96-well in 5% CO2 incubator (37 ^o C) 1hr.

Day 1

[0345] Apply drug(s) of interest 24 hours after seeding.

[0346] Drugs should be diluted into phenol red free RPMI-1640 supplemented as above.

[0347] Working stock of drug dilution should be at 3x the desired final concentration.

[0348] All required volumes should be calculated based on adding 50µL/well, giving a final volume of 150µL/well post application.

Day 2

[0349] Apply EdU 48 hours after seeding.

[0350] Add 50µL/well complete RPMI-1640 with 40µM EdU to each well, giving a final concentration of 10µM EdU in each well, and a final volume of 200µL in each well.

[0351] Note: wells not receiving EdU treatment will need 50µL of complete media added to them as well.

Day 3

[0352] Perform alalmarBlue assay

[0353] Dilute alamarBlue 1:1 in complete RPMI-1640 media and add 20µl/well to all wells.

[0354] Incubate 37 ^o C (5% CO ₂ ) for exactly 180 minutes.

[0355] Immediately read on a Molecular Devices Flexstation III (or similar platereader) using alamarBlue Ex:Em settings of:

[0356] Excitation: 560 nm; Emission: 588 nm; Cutoff: 570 nm Bottom read [0357] Process for cell proliferation‘click chemistry’ and EdU detection

[0358] Determine the volume of fix solution based on 100 µL per well.

[0359] Prepare fix solution by mixing 37% formaldehyde solution 1:10 with 1X PBS) (prepare fresh every day).

[0360] Remove medium from wells to be fixed.

[0361] Gently wash 1x with100 µL of PBS.

[0362] Add 100 µl of fixation, incubate RT in dark 15-20 minutes.

[0363] Gently wash 2x with PBS:

[0364] First wash is quick, on and off with 100 µl of PBS

[0365] Second wash is longer, 5-10 minutes RT in dark with 200 µl PBS

[0366] NOTE: Plate can be stored at this point by adding 100 µl of PBS to each well and placing in the dark at 4 ^o C. PBS used for storage must be removed before beginning permeabilization process.

[0367] Determine volume of permeabilization solution based on 100 µl per well.

[0368] Prepare 0.1% Triton X-100 in PBS permeabilization solution and filter through a 1 µm syringe filter.

[0369] Add 100 µl of permeabilization buffer to each well and incubate 15-20 minutes at RT in dark.

[0370] Gently wash 2x with PBS as above.

[0371] NOTE: Keep the last wash on and the plate in the dark until the click chemistry reagent cocktail is ready.

[0372] Determine total volume of click chemistry reaction cocktail based on 50 µl per well.

[0373] Prepare cocktail by combining, in this order:

[0374] PBS – 888 µL

[0375] CuSO ₄ – 10 µL

[0376] Azide-dye– 2 µL

[0377] Ascorbate– 100 µL

[0378] NOTE: 1mL of cocktail is good for 20 wells. Cocktail is good for 24 hours stored at 4 ^o C. [0379] Remove PBS, then add 50 µL of cocktail to each well and incubate at RT in dark for 30-60 minutes.

[0380] During click reaction incubation, determine volume of nuclear staining solution required (50 µL per well), and prepare 1x DAPI solution by diluting DAPI 1:1000 in PBS.

[0381] Gently wash 2x with PBS as above.

[0382] Add 50 µL of nuclear staining solution to each well and incubate at RT in the dark for 15 minutes.

[0383] Gently wash 2x with PBS as above

[0384] Add 50 µl of PBS before analysis and keep in dark for a minimum of 30 minutes before analysis.

Standard Conditions:

[0385] Cell density = 1 to 10 x10 ⁴ tumor cells / well

[0386] The results of the Standard 2D tissue culture assay are provided in Table 4 below.

TABLE 4: Standard 2D Tissue Culture Assay results

*IC50 Readings for AlamarBlue Shown Above EXAMPLE 5

MAXIMUM TOLERATED DOSE (MTD) ASSAY IN TUMOR-BEARING MICE [0387] In a first Maximum Tolerated Dose (MTD) study, mice bearing the MDA- MB-231 tumor line were orally (p.o) dosed at 1, 3, 10, 30, and 100 mg/kg of (Z)-3-(4- hydroxyphenyl)-5-((5-nitrofuran-2-yl)methylene)thiazolidine- 2,4-dione (1) once daily (Q.D.) over ten days. Administration of compound 1 showed efficacy in inhibiting the growth of large tumors (approximately 500 mm ³ starting size). Doses of 3, 10, 30, and 100 mg/kg of compound 1 exhibited tumor growth inhibition (TGI). The 3 mg/kg dose exhibited greater than 40% tumor growth inhibition relative to control, while the 100 mg/kg dose exhibited greater than 60% tumor growth inhibition relative to control. The maximum tolerated dose was not reached at the highest administered dose of 100 mg/kg p.o. Q.D. for 10 days.

[0388] In a second MTD study, mice bearing the MDA-MB-231 tumor line were orally (p.o) dosed at 0, 3, 10, 30, 100, and 300 mg/kg of (Z)-3-(4-hydroxyphenyl)-5-((5- nitrofuran-2-yl)methylene)thiazolidine-2,4-dione (1) once daily (Q.D.) over ten days. Administration of compound 1 showed efficacy in inhibiting the growth of standard size tumors (approximately 123 mm ³ starting size). Doses of 3, 10, 30, 100, and 300 mg/kg of compound 1 exhibited tumor growth inhibition (TGI). The maximum tolerated dose was not reached at the highest administered dose of 300 mg/kg p.o. Q.D. for 20 days. EXAMPLE 6

DOWNREGULATION OF pSTAT3 ^Y705 IN TUMOR CELLS Day 0:

[0389] Plate Tumor cells (MCF-7, BT-474, SK-BR-3) at 7000 cells per well in Tissue Culture treated Perkin Elmer Cell Carrier 96 well plates (Culture Media: RPMI-1640 + 10% FBS, 6 mM Glucose, 100 U/mL Penicillin, 100 ^g/mL Streptomycin, 2 mM L- glutamine). Cells were allowed to expand in culture for 48 hours. Day 2: Remove Media and Treat Tumor Cells

[0390] Remove medium, briefly wash and add 50 ml RPMI medium. [0391] Except for control wells, add drugs at 2X final concentration for 30 min before adding RPMI medium containing 100 ng/mL rh-IL6 (HumanZyme).

[0392] Final Drug Dose Range of AT-S-977.12: 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078 µM

[0393] Incubate cell cultures for 30 minutes

[0394] Fix for 10 minutes in 3.7% formaldehyde, permeablize using ice-cold methanol and stain with anti-pSTAT3 (Y705) mAB

Day 3: Finalize Immunofluorescent Staining Process and Image

[0395] Compete the staining with secondary Ab, followed by DAPI

[0396] High Content System (HCS) analysis (ImageXpress Micro)

[0397] The Compound 1 dose response as a percentage of control was measured for MCF-7, BT-474, and SK-BR-3 cells were measured via two independent experiments in triplicate. The data is summarized in Table 5 and Figures 2A-2C. TABLE 5: Downregulation of pSTAT3 ^Y705 results

*dose response as a percentage of control EXAMPLE 7

DRUG-INDUCED TRANSIENT MITOCHONDRIA-SOX INDUCTION [0398] MitoSOX was purchased from ThermoFisher Scientific (Catalog No. M36008) and was resuspended directly prior to use in DMSO, as per manufacturer’s instructions. Day 0: Plating of Cells [0399] Cells should be in exponential growth phase in standard 2D culture. Using standard cell culture technique, cells are trypsinized, pelleted, resuspended in cell culture media and counted as per SOP-11-022. Cells were plated in a Perkin Elmer Cell Carrier Ultra plate and allowed to expand in culture for 48 hours. Day 2: Experiment: sequential kinetic high content imaging

[0400] Immediately prior to use, thaw and resuspend MitoSOX stock in 13 µl DMSO to make a 5 mM stock, as per manufacturer’s instruction.

[0401] Make MitoSOX pre-loading solution into enough HBSS buffer to add 100 µl per well of experiment:

[0402] Dilute MitoSOX to 2 µM and counterstain Hoechst 33342 to 1:2000 (v:v)

[0403] MitoSOX: [2 uM x total volume desired in µl] = [5000 µM stock x Volume of MitoSOX stock in µl]

[0404] Add Hoechst 33342 stock 1:2000 (v:v)

[0405] For a negative MitoSOX control, add into HBSS the Hoechst counterstain 1:2000 (v:v).

[0406] Add 100 ul of either the MitoSOX + Hoechst pre-loading buffer or 100 µL of Hoechst only pre-loading buffer to the appropriate wells in the 96 well plate.

[0407] Incubate for 10 minutes in the incubator.

[0408] While incubating cells with the pre-loading buffers, make experimental drug solutions to add. These can be in either phenol red free media or in HBSS.

[0409] At the end of the 10 minutes pre-loading period, remove the buffer from the wells.

[0410] Carefully wash excess MitoSOX and/or Hoechst with 1-2 washes of HBSS.

[0411] Add experimental drugs to wells.

[0412] Immediately place in High Content Imager, and take sequential images using the DAPI/Hoechst channel (Ex/Em: ~350/470) for the counterstain, and either the FITC (Ex/Em: 488/525) or Cy3 (Ex/Em: 532/592) filters for the MitoSOX signal (Ex/Em: 510/590). The drug-induced Transient Mitochondria-SOX induction data is shown in Figure 1. EXAMPLE 8

NUCLEAR STAT1 ^Y701 IN HeLa CELLS FOLLOWING IFN-GAMMA EXPOSURE [0413] Cells were counted using TC10 Automated cell counter (Bio-Rad) and plate at 7,000 cells/well in complete medium in a 96-well plate (avoid plating in the outer wells to prevent any cell stress or dehydration). After 48 hours in culture (based on cell health and number), the culture medium was removed and 100 µl RPMI1640 without phenol red was added to wash the cells briefly. Dilute drugs (50 µl/well) in RPMI1640 without phenol red and incubated at 37 ºC for 30 min. Add 2X doses of drugs (2 µM, in 50 µl/well for 30 min followed by the addition of (a) IFN- γ, 10 ng/ml, final concentration (20 ng/ml, in 50 µl RPMI without phenol red) for 30 min before fixation.

Fixation

[0414] After the drug treatment, replace the medium with 100 µl/well, 3.7 % formaldehyde and incubate at room temperature for 10 min.

Permeabilization

[0415] Remove fixative and wash the cells twice with PBS followed by permeabilization using 100% ice-cold methanol (100 µl/well) at 4 ºC for 30 min.

Blocking

[0416] Wash permeabilized cells twice with PBS and block with 5% BSA, 0.1% Tween-20/PBS (100 µl/well) for 30 min at room temperature.

Incubation with primary antibody

[0417] After blocking, wash the cells 2 times with 0.1% Tween-20/PBS. Dilute primary antibody (Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb #7649, Cell Signalling) at 1:100 v/v in 1% BSA, 0.1% Tween-20/PBS and incubate over wells (50 µl/well) overnight at 4 ºC.

Incubation with secondary antibody

[0418] Wash off primary antibody and wash the cells twice with 0.1% Tween- 20/PBS (100 µl/well). Dilute secondary antibody (Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414; Cell Signalling at 1:500 (v/v) in PBS/0/.1% Tween- 20) and incubate at room temperature for 1 hour. Wash

[0419] Wash 2 times with 100 µl/well 0.1% Tween-20/PBS before DAPI staining.

DAPI staining

[0420] Add 100 µL of DAPI (1:1000 v/v) and incubate for 10 minutes.

Reading using High Content Imager

[0421] After washing off DAPI using 100 µL PBS, twice, read the plates using High Content Imager with saved settings for checking pSTAT1 nuclear localization. Figure 3 shows that Compound 1 does not inhibit pSTAT1 ^Y701 in HeLa cells. EXAMPLE 9

KINASE ACTIVITY [0422] The KiNativ ^TM Kinome Screen screened kinase inhibition following exposure of a 1 µM of either Compound 1, Nifuroxazide, and Ruxolitinib. Compound 1 displayed no kinase activity at doses of 0.1 µM, 1.0 µM, and 10 µM. Table 6 shows inhibition data for the top 20 kinases as ranked by ruxolitinib. Unlike ruxolitinib, Compound 1 and Nifuroxazide are not kinase inhibitors. TABLE 6: Kinase Activity

*dose response as a percentage of control EXAMPLE 10

IN VIVO ONCOLOGY DATA [0423] In vivo oncology data was collected in CT26 athymic nude Balb/c (Immune deficient) and CT26 Balb/c (Immune Competent) mice. No activity or toxicity was found for any drug or drug combination in athymic nude Balb/c mice (Figure 4A-4C). Administration of Compound 1 to CT26 Balb/c (Immune Competent) mice showed tumor growth inhibition (TGI), with a 24% TGI observed upon administration of a 1 mg/kg dose of Compound 1 as compared to control, and a 58% TGI observed upon administration of a 30 mg/kg dose of Compound 1 as compared to control after 20 days (Figure 5). Administration of Compound 1 in combination with either an anti-PD-1 or anti-CTLA-4 monoclonal antibody to CT26 Balb/c (Immune Competent) mice resulted in tumor growth inhibition, with a 26% TGI observed upon administration of anti-PD-1 and Compound 1, and a 99.9% TGI observed upon administration of anti-CTLA-4 monoclonal antibody and Compound 1 after 20 days (Figure 6). Administration of anti-CTLA-4 monoclonal antibody resulted in Immune-related adverse event at day 11, which resulted in the discontinuation of anti-CTLA- mAb administration in the anti-CTLA-4 monoclonal antibody and Compound 1 combination after day 11. [0424] Although the foregoing has been described in some detail by way of illustrations and examples for purposes of clarity and understanding, it will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present disclosure. Therefore, it should be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure, but rather to also cover all modification and alternatives coming with the true scope and spirit of the disclosure.