[0001] NITROALKENE TOCOPHEROLS AND ANALOGS THEREOF FOR USE IN THE TREATMENT AND PREVENTION OF INFLAMMATION RELATED CONDITIONS

Background

[0002] Atherosclerosis is a chronic, progressive disease of the arterial wall and a leading cause of death worldwide. This disease is the main cause of heart attack and stroke and is responsible for almost 50% of all mortality in the USA and Europe (1). Atherosclerosis affects main arteries of the organism and its lesion, the atheromatous plaque, is a local, elevated formation localized at the intima layer of the artery wall. The atheromatous plaque is composed of cells (smooth muscle, endothelial and foam cells and leukocytes), and an adipose nucleus (cholesterol, cholesterol esters and its oxidation products and cellular debris) surrounded by a fibrous cap (2). Atherosclerotic lesions result from an excessive, inflammatory-fibroproliferative response to various forms of aggravation to the endothelium and smooth muscle of the artery wall. A large number of growth factors, cytokines and vasoregulatory molecules participate in this process (3).

[0003] There are different, well-known, major cardiovascular risk factors that affect the development of the disease (plasma Low Density Lipoprotein (LDL)-cholesterol level, hypertension, diabetes and smoking) (3, 4). Among all the factors, the most remarkable is the relationship between high levels of plasma LDL and the risk of cardiovascular disease (5). It has been postulated that atherogenesis begins with the recruitment of inflammatory cells to the intima layer of the artery wall, generation of oxidative inflammatory conditions that leads to oxidative modifications of LDL and endothelial dysfunction (7, 8). Activated endothelial cells express leukocyte adhesion molecules that capture blood monocytes. The blood monocytes express scavenger receptors which permit the uptake of modified/oxidized LDL particles, leading to foam cell formation. All these cells produce pro-inflammatory mediators and reactive oxygen species that amplify local inflammation and promote lesion progression (7, 8).

Accordingly, atherosclerosis is actually viewed as a chronic low-grade inflammatory process of the vessel wall that initiates and promotes lesion development.

[0004] With this intersection of inflammation and lipid metabolism, which modulates

atherosclerosis, understanding the molecular mechanisms responsible for inflammatory reactions during atherogenesis will facilitate the development of novel anti-inflammatory therapeutic strategies to control, treat and prevent atherosclerosis (8, 9). [0005] Studies have shown that endogenous electrophilic unsaturated-nitrated fatty acids, including nitro-linoleic acid (LN0 ₂ ) and nitro-oleic acid (OA-N0 ₂ ), can mediate antiinflammatory and pro-survival signaling reactions (10). The basis for the mediation is mainly produced because the presence of the nitro group on the double bond imparts a strong electrophilic character to the β-carbon adjacent to the nitroalkene, which promotes covalent bonding with nucleophiles in proteins (thiols and histidine residues) and low molecular weight molecules via Michael addition reactions (11).

[0006] Thus, biological electrophilic, oxidized or nitrated fatty acids, which include N0 ₂ -FA, cyclooxygenase-derived 15-deoxy-A12,14-PGJ2, a variety of isoprostane derivatives and lipoxygenase-derived α,β-unsaturated ketones, are emerging as mediators protecting against xenobiotic and oxidant injury. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2)/Keapl (Kelch-like ECH-associating protein) pathway mediates phase 2 gene activation (12). Under normal conditions, Nrf2 localizes to the cytoplasmic supressor protein Keapl which has several critical cysteine residues that serve as sensors to environmental stresses such as reactive oxygen species (ROS) and electrophiles (12, 13). Keapl cysteines are oxidized or alkylated, causing a conformational change and liberating Nrf2 to translocate to the nucleus, bind to the cis-acting DNA regulatory antioxidant response element (ARE) and thereby

transactivating Nrf2-dependent gene transcription (12, 13). This includes enzymes involved in glutathione (GSH) metabolism such as the subunits of the rate-limiting enzyme of glutathione synthesis, glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit genes. Also NAD(P)H:quinone oxidoreductase-1 (NQOl), which not only detoxifies xenobiotic quinones, but also reduces antioxidants vitamin E and coenzyme Q10 to their active form, is a Nrf2 target gene.

[0007] In addition, HO-1 has been shown to be positively regulated by Nrf2 (14, 15). This widespread mechanism protects against metabolic and inflammatory stress (14, 16, 17). It is interesting to note that electrophilic nitro-fatty acids activate NRF2 by a KEAPl cysteine 151- independent mechanism (18). Actually, nitrated oleic acid, one of the endogenous nitroalkenes, is a Cys(15 l)-independent Nrf2 activator, which in turn can influence the pattern of gene expression and therapeutic actions of nitroalkenes (18).

[0008] Heme Oxygenase- 1 (HO-1) also plays a central role in vascular inflammatory signaling and mediates a protective response to inflammatory stresses such as atherosclerosis, vascular restenosis and kidney diseases including transplant rejection (19). Heme oxygenase 1 catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide (CO). CO has been shown to display diverse, adaptive biological properties, including anti-inflammatory, anti-apoptotic, and vasodilatory actions (20).

[0009] Nitrated fatty acids have also been shown to be activators of peroxisome proliferator- activated receptor gamma (PPARy). PPARy is established as a master regulator of metabolism, inflammation, adipogenesis, and insulin sensitization (21). High, non-physiological

concentrations of native fatty acids (N50 μΜ), prostaglandin metabolites, and oxidized fatty acid derivatives are able to activate PPARy, a, and δ (20, 22). Fatty acids containing an α-β- unsaturated ketone as a core structural element, such as 15d-PGJ ₂ , also activate PPARy (23). Docking of 15d-PGJ ₂ to the ligand binding domain (LBD) shows that it is not sufficient to activate the receptor; rather, a covalent Michael addition reaction (locking reaction) is required for activation (20). The PPARy receptor contains a critical thiol (Cys285) in the LBD, with covalent modification of this highly conserved Cys285 by thiol-reactive compounds sufficient to induce partial receptor activation (24). N0 ₂ -FAs and keto-fatty acid derivatives have high binding affinities for PPAR isotypes, being PPARy the most robustly-activated receptor (13, 20, 25). The mechanism by which LN02 activates PPARy has been determined with recent solution of the crystal structure of PPARy having LN02 occupancy in the LBD (26, 27). Differential conformational changes to PPARy resulting from this unique endogenous ligand have the capacity to impart unique specificity to the downstream signaling events resulting from PPARy activation (20, 27). Interestingly, PPARy activation skews human monocytes toward an antiinflammatory M2 phenotype (28), another possible mechanism to further explain the antiinflammatory, anti-atherogenic properties of endogenous nitroalkenes.

[0010] However, in vivo studies of nitrated oleic acid (18: 1-N0 ₂ ) metabolism showed that nitrated oleic acid undergoes a rapid and substantial modification that affects subsequent chemical reactivity and signaling actions (29). More specifically, the results of the study showed the 18:1-N0 ₂ suffers rapid but reversible adduction to plasma thiol-containing thiols and GSH. Furthermore, a significant proportion of 18: 1-N0 ₂ and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. The rapid saturation of the double bond decreases the electrophilic character of the molecule and may consequently affect the potency (29). [0011] The study also showed that nitro-oleic acid is metabolized by the β-oxidation pathways. As a result, the β-oxidized metabolite will be less hydrophobic and this will not only influence partitioning between hydrophobic and hydrophilic compartments and consequent tissue distribution, but can also affect chemical reactivity and pharmacological profiles by altering the specificity of the nitroalkenes to the biologically relevant targets.

[0012] In the case of the present invention, nitroalkene tocopherols and analogs thereof, exhibit comparable potency as anti-inflammatory nitrated fatty acids. Furthermore, the present invention mimics the transport processes of other lipid molecules in vivo and is closely related to lipoprotein homeostasis and metabolism which control intestinal absorption, traffic through the vascular compartment, and cellular uptake (30).

[0013] For example, after feeding the majority of the dietary vitamin E is incorporated into chylomicrons and ultimately reaches the liver parenchymal cells transported in remnant lipoprotein particles. The liver is a major storage site of vitamin E, accounting for one-third of the total body content of this vitamin (31, 32). After hepatic uptake, the a-tocopherol form of vitamin E is preferentially secreted into the circulation via the VLDL-LDL system by Alpha- Tocopherol Transfer Protein (a-TTP). The a-TTP is a small cytoplasmic hepatic protein with differential affinity for various vitamin E forms, and is responsible for the biodiscrimination process underlying the selective secretion of a-tocopherol from the liver into plasma LDL (30). Thus providing a mechanism for incorporation of a nitroalkene vitamin E hybrid into LDL particles. After incorporating the nitroalkene vitamin E, the LDL protects the compound from rapid β-oxidation, and transports the nitroalkene vitamin E to the specific pharmacological target.

[0014] Therefore, compared with the N02-fatty acid derivatives, the nitroalkene-a-tocopherol (NA-a-TOH or NATOH) analogs have several advantages: (i) their metabolism is dependent on the structure of the tocopherol; (ii) they are not β-oxidized because the nitroalkene is not attached to a fatty acid; and (iii) they are most likely not metabolized by the nitroalkene reductase

(because of the distance between the nitroalkene and the terminal carbon (33). Furthermore, its hydrophobicity and cellular localization can be regulated by modifying the length of the isoprenoid chain.

Summary

[0015] Within the scope of the present invention is a new pharmacological strategy for the treatment of conditions associated with chronic inflammation. In one embodiment, the present invention includes the treatment of atherosclerosis based on the main pathogenic role that low density lipoproteins and chronic inflammation play in atherogenesis (6, 8, 34). This

pharmacological approach implies that the hybrid compound would be selectively incorporated into the lipoproteins particles during the normal metabolism and due to the presence of the chromanol structure of tocopherol. Once the nitroalkene-vitamin E-analog is incorporated into the LDL, this lipoprotein will carry it all over the body, including the atherosclerotic lesions, where the hybrid compound will be able to exert the potent anti-inflammatory and antiatherogenic properties similar to nitrated fatty acids but without the disadvantages of β-oxidation and lack of control over the targeting and localization of the compound. Thus, during normal metabolism, the LDL particle will be used as a carrier of the nitroalkene-hybrid compound to the lesion sites. Included within the scope of the present invention is a novel hybrid compound analog of Vitamin E (tocopherol) that includes an electrophilic nitroalkene functional group.

[0016] In another embodiment the present invention includes a novel hybrid compound analog of Vitamin E that includes an electrophilic nitroalkene functional group (NA-a-TOH). This pharmacological approach implies that the hybrid compound would be selectively incorporated into the lipoproteins particles during the normal metabolism and due to the presence of the chromanol structure of tocopherol.

[0017] In another embodiment the present invention includes novel hybrid compound analogs of trolox that include an electrophilic nitroalkene functional group such as for example a trolox methyl ester that includes an electrophilic nitroalkenes functional group (NA-Trolox-Me or NA- Tx-Me). This pharmacological approach implies that the hybrid compound would be selectively incorporated into the lipoproteins particles during the normal metabolism and due to the presence of the chromanol structure of trolox.

[0018] Further embodiments within the scope of the present invention include trolox derivatives and metabolites that are not incorporated into LDL particles and still serve as anti-inflammatory agents. Accordingly, this embodiment includes trolox molecules include hydrophilic functional groups in lieu of alkyl esthers, alkyls, and other hydrophobic groups.

[0019] In another embodiment, the R ¹ group of the compound represented by Formula I comprises lipophilic groups.

[0020] In another embodiment, the R ¹ group of the compound represented by Formula I comprises amphiphilic groups. [0021] Another embodiment of the present invention includes a class of hybrid compounds containing chromanol structures within tocopherol motif based compounds that include a nitroalkene or nitro functional group.

[0022] In yet another embodiment, a class of hybrid compounds comprised of fatty acids containing chromanol structures, wherein the chromanol containing fatty acids have a nitro group or nitro alkene group.

[0023] In another embodiment the present invention includes pharmaceutical compositions of the hybrid compound.

[0024] In yet another embodiment the present invention contemplates a method of making the hybrid compound analog includes an easy reaction to modify the length of the desired isoprenoid chain to control hydrophobicity and cellular localization.

[0025] In another embodiment, the present invention contemplates a method of controlling the hydrophobicity of the molecule by increasing or decreasing the length of the nitroalkene, wherein the double bond is further or closer to the chromanol structure.

[0026] A method of treating inflammation related conditions comprising the steps of

administering to a subject in need thereof a pharmaceutical composition comprising a nitroalkene tocopherol analog or pharmaceutically acceptable salt thereof.

[0027] In another embodiment, the method of treating inflammation related conditions comprises the steps of drawing a first sample from the subject before administering the pharmaceutical composition, administering the pharmaceutical composition to the subject in need thereof, drawing a second sample from the subject at a reasonable time after administering the pharmaceutical composition to the subject, testing the first and second samples for cell signaling factors, comparing results from testing the first and second samples, and repeating the administering step with the proviso that if the cell signaling factors show effective protein expression, mR A transcription, or cytokine expression as compared to the first sample, repeating the administering step is not necessary.

[0028] The methods, systems, and apparatuses are set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the methods, apparatuses, and systems. The advantages of the methods, apparatuses, and systems will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the methods, apparatuses, and systems, as claimed.

Brief Description Of The Drawings

[0029] FIG. 1 illustrates the relative expression of the HO-1 protein after exposure to

NA-Trolox-ME.

[0030] FIG. 2 illustrates the relative expression of the GCLM protein after exposure to

N-Trolox-ME.

[0031] FIG. 3 illustrates the relative quantity of mRNA transcription for the HO-1, GCLM, and NQOl proteins after raw cells were treated with NA-Trolox-ME.

[0032] FIG. 4 illustrates the concentration of cytokine TNF a after activated cells were treated with NA-Trolox-ME.

[0033] FIG. 5 illustrates the concentration of cytokine MCP-1 after activated cells were treated with NA-Trolox-ME.

[0034] FIG. 6 illustrates the concentration of cytokine IL-6 after activated cells were treated with NA-Trolox-ME.

[0035] FIG. 7 illustrates the relative quantity of RNA transcription for HO-1 after in vivo administration of NA-a-TOH and NA-Trolox-ME.

[0036] FIG. 8 illustrates the relative quantity of RNA transcription for GCLM-1 after in vivo administration of NA-a-TOH and NA-Trolox-ME.

[0037] FIG. 9 illustrates the relative quantity of RNA transcription for NQOl after in vivo administration of NA-a-TOH and NA-Trolox-ME.

[0038] FIG. 10 illustrates the electrophilic character of NA-a-TOH

[0039] FIG. 11 illustrates shows NA-a-TOH as a potent electrophilic mediator of reversible nitroalkylation reactions with both BME and GSH.

[0040] FIG. 12 is a general chemical structure of an embodiment of the invention.

[0041] FIG. 13 is a general chemical structure of another embodiment of the invention.

[0042] FIG. 14 is a general chemical structure of another embodiment of the invention.

Detailed Description

[0043] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. [0044] It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0045] It must also be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.

[0046] As used herein, the term "about" means plus or minus 5% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%.

[0047] "Administering" when used in conjunction with a therapeutic means to administer a therapeutic directly to a subject, whereby the agent positively impacts the target.

"Administering" a composition may be accomplished by, for example, parenterally, oral administration, topical administration, or by these methods in combination with other known techniques. Such combination techniques include heating, radiation, ultrasound and the use of delivery agents. When a compound is provided in combination with one or more other active agents (e.g. other anti-atherosclerotic agents such as the class of statins), "administration" and its variants are each understood to include concurrent and sequential provision of the compound or salt and other agents.

[0048] By "pharmaceutically acceptable" it is meant the carrier, diluent, adjuvant, or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0049] "Composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such term in relation to "pharmaceutical composition" is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the

pharmaceutical compositions of the present invention encompass any composition made by admixing a compound o the present invention and a pharmaceutically acceptable carrier.

[0050] As used herein, the term "agent," "active agent," "therapeutic agent," or "therapeutic" means a compound or composition utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a patient. Furthermore, the term "agent," "active agent," "therapeutic agent," or "therapeutic" encompasses a combination of one or more of the compounds of the present invention.

[0051] A "therapeutically effective amount" or "effective amount" of a composition is a predetermined amount calculated to achieve the desired effect, i.e., to inhibit, block, or reverse the activation, migration, proliferation, alteration of cellular function, and to preserve the normal function of cells. The activity contemplated by the methods described herein includes both medical therapeutic and/or prophylactic treatment, as appropriate, and the compositions of the invention may be used to provide improvement in any of the conditions described, and in particular embodiments, the effective amount may 1) prevent the subject from experiencing one or more adverse effects associated with a administered agents, such as those used to diagnose, identify, and treat medical conditions, 2) reduce side effects experienced by the subject as a result of a medical therapy or reduce the side effects known to result from such therapies, and/or 3) eliminate side effects resulting from a medical treatment experienced by the subject prior to administration of the active agent or eliminate the side effects known to result from such treatment. It is also contemplated that the compositions described herein may be administered to healthy subjects or individuals not exhibiting symptoms but who may be at risk of developing a particular disorder. The specific dose of a compound administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, and the condition being treated. However, it will be understood that the chosen dosage ranges are not intended to limit the scope of the invention in any way. A therapeutically effective amount of compound of this invention is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the tissue.

[0052] The terms "treat," "treated," or "treating" as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or to obtain beneficial or desired clinical results. For the purposes of this invention, beneficial or desired results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder, or disease; stabilization (i.e., not worsening) of the state of the condition, disorder, or disease; delay in onset or slowing of the progression of the condition, disorder, or disease; amelioration of the condition, disorder, or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder, or disease. Treatment includes prolonging survival as compared to expected survival if not receiving treatment.

[0053] Among its many embodiments the present invention provides a compound of Formula I

[0054] or a pharmaceutically-acceptable salt thereof, wherein: R ¹ is selected from a methyl ester, an ethyl ester, a propyl ester, a butyl ester, a carboxyl, a phytanyl, or a phytenyl; and R ² is selected from a -CH ₃ , a Ci-C ₆ nitroalkene, a Ci-C ₆ cyanoalkene, a Ci-C ₆ conjugated nitroalkene, a Ci-C ₆ allyl ethyl ester, a Ci-C ₆ alpha-cyano enone, and a Ci-C ₆ alpha-cyano enoate.

[0055] Within Formula I there is a subclass of compounds of high interest represented by Formula II:

or a pharmaceutically-acceptable salt thereof, wherein: R ³ is N0 ₂ or -H; R ⁴ is N0 ₂ or -H; and R ⁵ is N0 ₂ or -H

[0056] Within Formula I there is a subclass of compounds of high interest represented by Formula III:

or a pharmaceutically-acceptable salt thereof, wherein: R ² is selected from a -CH ₃ , a Ci- C ₆ nitroalkene, a Ci-C ₆ cyanoalkene, a Ci-C ₆ conjugated nitroalkene, a Ci-C ₆ allyl ethyl ester, a Ci-C ₆ alpha-cyano enone, and a Ci-C ₆ alpha-cyano enoate.

[0057] Also included in the family of compounds of Formula I, Formula II, and Formula III are tautomers and stereoisomers thereof. In regard to stereoisomers, compounds of the present invention can possess one or more asymmetric carbon atoms and are thus capable of existing in the form of optical isomers as well as in the form of racemic or nonracemic mixtures thereof. Accordingly, some of the compounds of this invention may be present in racemic mixtures which are also included in this invention.

[0058] In one embodiment, the preferred compound of the present invention of Formula I presents absolute configuration R in carbon 2. In one embodiment, the preferred compound of the present invention of Formula II presents absolute configuration of R in carbon 2. In one embodiment, the preferred compound of the present invention of Formula III presents absolute configuration of R in the stereocenters 2, 4', and 8'.

[0059] The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example by formation of diastereoisomeric salts by treatment with an optically active base and then separation of the mixture of diastereoisomers by crystallization, followed by liberation of the optically active bases from these salts. Examples of appropriate bases are brucine, strychnine, dehydroabietylamine, quinine, cinchonidine, ephedrine, a- methylbenzylamine, amphetamine, deoxyphedrine, chloramphenicol intermediate, 2-amino-l- butanol, and l-(l-maphtyhl)ethylamine. Another method calls for chiral separation of the enantiomers with the use of a chiral chromatography column optimized to maximize the separation of the enantiomers. Optimization of the chromatographic method of chiral resolution is routine for one of ordinary skill in the art. Yet another method for isolating optical isomers is by distillation, crystallization or sublimation if a physical property of the enantiomers is different. The optically active compounds of Formula I, Formula II, and Formula III can also be obtained by utilizing optically active starting materials. The isomers may be in the form of a free acid, a free base, an ester or a salt.

[0060] In one embodiment of the present invention, the tautomer of the chroman structure is in the keto or enol form.

[0061] Also included in the family of compounds of Formula I, Formula II, and Formula III and their tautomers and stereoisomers are the pharmaceutically-acceptable salts thereof. The term

"pharmaceutically-acceptable salts" embraces salts commonly used to form alkali metal salts and to form additional salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically-acceptable acid addition salts of compounds of Formula I, Formula II, and Formula III may prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may include aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids. Examples of such organic acids include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, 4-hydrobenzoic, phylacetic, mandelic, embonic, methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2- hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohyexylaminosuflonic, stearic, algenic, β- hydrobutyric, galactaric and galacturnoic acid.

[0062] Suitable pharmaceutically-acceptable base addition salts of compounds of Formula I, Formula II, and Formula III include metallic salts, such as salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or salts made from organic bases including primary, secondary and tertiary amines, substituted amines including cyclic amines, such as caffeine, arginine, diethylamine, N-ethyl piperidine, histidine, glucamine, isopropylamine, lysine, morpholine, N-ethyl morpholine, piperazine, triethylamine, trimethylamine.

[0063] All the listed salts of the corresponding compound of the invention may be prepared by conventional means known to one of ordinary skill in the art. One example of a conventional method of salt formation is by reacting the appropriate acid or base with the compounds of Formula I or Formula II at various mole ratios. Another method is by using different mole ratios of the appropriate acid or base in various solvent systems to control the concentration of the dissociated species of the compounds of Formula I or Formula II to maximize salt formation. The present invention also contemplates crystalline forms of the salts described herein.

[0064] Crystalline forms of the compounds of Formula I, Formula II, and Formula III may also include but are not limited to hydrates, solvates, and co-crystals. Crystalline solvates include solvents including but not limited to the following: MeOH, EtOH, AcOH, EtOEt, AcOEt, acetone, DMSO, DMF, MeCN, CH ₂ C1 ₂ , CHC1 ₃ , CC1 ₄ , dioxane, THF, benzene, toluene, p-xylene, and hexane.

[0065] Crystalline hydrates and solvates may be stoichiometric as according to the mole ratio of the water or organic solvent molecule to the compound or salt thereof. The crystalline hydrate may also be non-stoichiometric depending on the conditions of the unit cell which result in a thermodynamically or kinetically stable crystal. Crystalline salts and co-crystals may also be stoichiometric or non-stoichiometric for reasons stated above. One of skill in the art of crystallography understands that the components in the unit cell of a crystal may or may not be stoichiometric depending on the conditions which stabilize the crystal.

[0066] Administration

[0067] The compounds of Formula I, Formula II, and Formula III can be administered to a patient afflicted with a chronic disease wherein chronic inflammation is a root cause. Examples of such disorders associated with inflammation include but are not limited to the following: atherosclerosis; acne vulgaris; asthma; autoimmune diseases; celiac disease; diabetes;

glomerulonephritis; high blood pressure; hypersensitivities; inflammatory bowel diseases; pelvic inflammatory disease; ischemia-reperfusion injury; rheumatoid arthritis; sarcoidosis; sepsis; transplant rejection; vasculitis; and ventilator-induced lung injury. [0068] The compounds and pharmaceutically-acceptable salts thereof can be administered by means that produces contact of the active agent with the agent's site of action. They can be administered by conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but typically are administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

[0069] Compounds can be administered by one or more ways. For example, the following routes may be utilized: parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation, or rectally, in the form of a unit dosage of a pharmaceutical composition containing an effective amount of the compound an pharmaceutically-acceptable carrier, adjuvants, vehicles and diluents. Liquid preparations suitable for oral administration (e.g. suspensions, syrups, elixirs and other similar liquids) can employ media such as water, glycols, oils, alcohols, and the like. Solid preparations suitable for oral administration (e.g. powders, pills, capsules and tablets) can employ solid excipients such as starches, sugars, kaolin, lubricants, binders, disintegrating agents, antioxidants and the like. Parenteral compositions typically employ sterile water as a carrier and optionally other ingredients, such as solubility aids. Injectable solutions can be prepared, for example, using a carrier comprising a saline solution, a glucose solution or a solution containing a mixture of saline and glucose. Further guidance for methods suitable for use in preparing pharmaceutical compositions is provided in Remington: The Science and Practice of Pharmacy, 21 ^st edition (Lippincott Williams & Wilkins, 2006).

[0070] Therapeutic compounds can be administered orally in a dosage range of 0.001 to 1000 mg/kg of mammal (e.g. human) body weight per day in a single dose or in divided doses. One dosage range is 0.01 to 500 mg/kg body weight per day orally in a single dose or in divided doses. For oral administration, the compositions can be provided in the form of tablets or capsules containing 1.0 to 500mg of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, and 750 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. In view of the factors affecting the specific dose level and frequency it is contemplated that the dose frequency can range from twice a day to monthly dosages. The preferred dose frequency ranges from twice a day to every two weeks. A more preferred dose frequency ranges from once a day to weekly. A most preferred dose frequency ranges from once a day to every other day.

[0071] When administered parenterally in a dosage range of 0.001 to 1000 mg/kg of mammal (e.g. human) body weight per day in a single dose or in divided doses. One dosage range is 0.01 to 500 mg/kg body weight per day orally in a single dose or in divided doses. Unit doses can contain, for example, from about 1 mg to about 200 g of the active agent. Thus, ampoules for injection can contain, for example, from about 1 mg to about 200 g. Preferred dosages include 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, and 750 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. In view of the factors affecting the specific dose level and frequency it is contemplated that the dose frequency can range from twice a day to monthly dosages. The preferred dose frequency ranges from twice a day to every two weeks. A more preferred dose frequency ranges from once a day to weekly. A most preferred dose frequency ranges from once a day to every other day. In the methods of various embodiments,

pharmaceutical compositions including the active agent can be administered to a subject in an "effective amount."

[0072] When administered intravenously, the daily dose, can for example, be in the range from about 0.01 mg/kg body weight to about 20 mg/kg body weight, in another embodiment 0.25 mg/kg body weight to about 10 mg/kg body weight, in another embodiment from about 0.4 mg/kg body weight to about 5 mg/kg bodyweight. The dose can be administered as an infusion of about 10 ng/kg body weight to 2 μg/kg body weight per minute. Infusion fluids suitable for this purpose can contain, for example, from about 0.1 ng to about 10 mg per milliliter, in another embodiment from about 1 mg to about 200 mg per milliliter. Unit doses can contain, for example, from about 1 mg to about 200 g of the active agent. Thus, ampoules for infusion can contain, for example, from about 1 mg to about 200 g. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. In view of the factors affecting the specific dose level and frequency it is contemplated that the dose frequency can range from twice a day to monthly dosages. The preferred dose frequency ranges from twice a day to every two weeks. A more preferred dose frequency ranges from once a day to weekly. A most preferred dose frequency ranges from once a day to every other day. For each administration of a dose, the length of time for administration of the dose by infusion may range from about 1 minute to about 1 day. The preferred infusion time is from about 5 minutes to 12 hour. The more preferred infusion time is from about 10 minutes to 1 hour. The most preferred infusion time is from about 15 minutes to 30 minutes. In the methods of various embodiments, pharmaceutical compositions including the active agent can be administered to a subject in an "effective amount."

Compositions

[0073] Some embodiments of the present invention include a pharmaceutical composition comprising an effective amount of the active agent and a pharmaceutically acceptable carrier. Other embodiments include a pharmaceutical composition comprising an effective amount of pharmaceutically-acceptable salts of the active agent. Other embodiments include a

pharmaceutical composition comprising an effective amount of pharmaceutically-acceptable salts of active agent and a pharmaceutically-acceptable carrier.

[0074] In yet other embodiments, the active agent may be combined with one or more secondary agents. Examples of secondary agents may vary depending upon the therapeutic or diagnostic agent administered to the patient and include, but are not limited to statins, beta-blockers, calcium antagonists, angiotensin-converting enzyme inhibitors, inhibitors of angiotensin II receptors (ATI inhibitors), diuretics, and anti-inflammatory agents such as but not limited to acetyl salicylic acid and omega- 3 fatty acids.

[0075] Embodiments of the invention also include gel capsules containing the active agent and, in some embodiments, one or more secondary agents. The gel capsules of embodiments may be in soft or hard gel capsule form and may include any number of layers. For example, the gel capsule may include the active agent encapsulated by a coating layer. In such embodiments, the active agent may make up the core of the capsule and may generally be from about 1% by weight to about 95% by weight of the total gel capsule. However, in some embodiments, the core may be from about 20% by weight to about 90% by weight of the total weight of the capsule. In other embodiments the core may be from about 35% to 75% by weight of the total weight of the capsule. In particular embodiments, the active agent may be mixed with one or more stabilizers such as, for example, antioxidants, vitamin E, vitamin C, β-carotene, wheat germ oil and the like, and in some embodiments the capsule may be combined with one or more solubilizers such as, for example, surfactants, hydrophilic or hydrophobic solvents, oils, or combinations thereof.

[0076] In other embodiments, monohydric alcohol including, for example, ethanol, isopropanol, t-butanol, a fatty alcohol, phenol, cresol, benzyl alcohol or a cycloalkyl alcohol, or monohydric alcohol esters of organic acids such as, for example, acetic acid, propionic acid, butyric acid, a fatty acid of 6-22 carbon atoms, bile acid, lactic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, and salicylic acid may be used as solubilizers. In certain embodiments, solubilizers in this group may include trialkyl citrates such as triethyl citrate, acetyltriethyl citrate, tributyl citrate, acetyltributyl citrate, and mixtures thereof; lower alcohol fatty acid esters such as ethyl oleate, ethyl linoleate, ethyl caprylate, ethyl caprate, isopropyl myristate, isopropyl palmitate and mixtures thereof and lactones ε-caprolactone, δ-valerolactone, β-butyrolactone, isomers thereof, and mixtures thereof.

[0077] In still other embodiments, the solubilizer may be a nitrogen-containing solvent such as, for example, acetonitrile, dimethylformamide, dimethylacetamide, N-alkylpyrrolidone, N- hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, and mixtures thereof wherein alkyl may be a C _1-12 branched or straight chain alkyl. In particular embodiments, nitrogen- containing solvents may include N-methyl 2-pyrrolidone, N-ethyl 2-pyrrolidone, or a mixture thereof. Alternatively, the nitrogen-containing solvent may be in the form of a polymer such as polyvinylpyrrolidone.

[0078] In yet other embodiments, solubilizers may include phospholipids such as

phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lecithins, lysolecithins, lysophosphatidylcholine, polyethylene glycolated

phospholipids/liysophospholipids, lecithins/lysolecithins and mixtures thereof.

[0079] In still other embodiments, glycerol acetates and acetylated glycerol fatty acid esters and glycerol fatty acid esters may be used as solubilizers. In such embodiments, glycerol acetates may include acetin, diacetin, triacetin, and mixtures thereof. Acetylated glycerol fatty acid esters may include acetylated monoglycerides, acetylated diglycerides, and mixtures thereof with a fatty acid component that may be about 6 to about 22 carbon atoms. Glycerol fatty acid ester may be a monoglyceride, diglyceride, triglyceride, medium chain monoglycerides with fatty acids having about 6-12 carbons, medium chain diglycerides with fatty acids having about 6-12 carbons, medium chain triglycerides with fatty acids having about 6-12 carbons, and mixtures thereof.

[0080] Further embodiments include solubilizers that may be propylene glycol esters or ethylene glycol esters. In such embodiments, propylene glycol esters may include, for example, propylene carbonate, propylene glycol monoacetate, propylene glycol diacetate, propylene glycol fatty acid esters, acetylated propylene glycol fatty acid esters, and mixtures thereof.

Alternatively, propylene glycol fatty acid esters may be a propylene glycol fatty acid monoester, propylene glycol fatty acid diester, or mixture thereof. In certain embodiments, propylene glycol ester may be propylene glycol monocaprylate, propylene glycol dicaprylate, propylene glycol dicaprate, propylene glycol dicaprylate/dicaprate, and mixtures thereof. Ethylene glycol esters may include monoethylene glycol monoacetates, diethylene glycol esters, polyethylene glycol esters, ethylene glycol monoacetates, ethylene glycol diacetates, ethylene glycol fatty acid monoesters, ethylene glycol fatty acid diesters, polyethylene glycol fatty acid monoesters, polyethylene glycol fatty acid diesters, and mixtures thereof. In such embodiments, the fatty acid may have about 6 to about 22 carbon atoms.

[0081] Hydrophilic solvents may also be utilized as solubilizers include, for example, alcohols, for example, water miscible alcohols, such as, ethanol or glycerol; glycols such as 1 ,2-propylene glycol; polyols such as a polyalkylene glycol, for example, polyethylene glycol. Alternatively, hydrophilic solvents may include N-alkylpyrolidones such as N-methylpyrolidone,

triethylcitrate, dimethylisosorbide, caprylic acid, or propylene carbonate.

[0082] The core comprised of an active agent may be coated with one or more coating layer. For example, in some embodiments, the gel capsule may include a water-soluble gel layer between the coating layer and the active agent. In other embodiments, the gel capsules may include a number of additional coatings on the capsules such as, for example, immediate release coatings, protective coatings, enteric or delayed release coatings, sustained release coatings, barrier coatings, and combinations thereof. In some embodiments, one or more secondary agent may be mixed with the active agent and/or be present in either a coating layer, a water-soluble gel layer, or an additional coating layer.

[0083] Additionally, in various embodiments, the core and/or one or more secondary agents of the invention may be formulated with one or more additional non-pharmaceutically active ingredients including, but not limited to, solubilizers, antioxidants, chelating agents, buffers, emulsifiers, thickening agents, dispersants, and preservatives. In some embodiments, the active agent may be encapsulated in a coating prepared from gelatin as described in U.S. Patent No. 6,531,150, which is hereby incorporated by reference in its entirety. The gelatin layer may further include one or more other non-gelatin protein and/or one or more polysaccharide such as, for example, albumin, pectin, guaran gum, carrageenan, agar, and the like, and/or one or more additive such as, for example, enteric materials, plasticizers, preservatives, and the like. Enteric materials used in embodiments of the invention include any material that does not dissolve in the stomach when the gel capsule is administered orally and include, but are not limited to, pectin, alginic acid, cellulose such as carboxyl methylcellulose, celluloseacetate phthalate, and the like, and Eudragit™, an acrylic copolymer. Without wishing to be bound by theory, the addition of an enteric coating may provide a means for masking the flavor of the active agent by limiting the release of the compounds to the stomach. Plasticizers may include polyhydric alcohols, such as sorbitol, glycerin, polyethylene glycol, and the like. In the embodiments described above, each coating layer may be from about 0.001 to about 5.00 mm or 0.01 to 1.00 mm thick.

[0084] The coatings of various embodiments may further include one or more film forming materials and/or binders and/or other conventional additives such as lubricants, fillers, antiadherents, antioxidants, buffers, solubilizers, dyes, chelating agents, disintegrants, and/or absorption enhancers. Surfactants may act as both solubilizers and absorption enhancers.

Additionally, coatings may be formulated for immediate release, delayed or enteric release, or sustained release in accordance with methods well known in the art. Conventional coating techniques are described, e.g., in Remington's Pharmaceutical Sciences, 18th Ed. (1990), hereby incorporated by reference. Additional coatings to be employed in accordance with the invention may include, but are not limited to, for example, one or more immediate release coatings, protective coatings, enteric or delayed release coatings, sustained release coatings, barrier coatings, and combinations thereof. In some embodiments, an immediate release coating may be used to improve product elegance as well as for a moisture barrier, and taste and odor masking. Rapid breakdown of the film in gastric media is important, leading to effective disintegration and dissolution.

[0085] Capsular materials (i.e., the compound containing core and/or one or more coating layers) may further include one or more preservatives, coloring and opacifying agents, flavorings and sweeteners, sugars, gastroresistant substances, or combinations thereof. Suitable preservative and colorant are known in the art and include, for example, benzoic acid, para-oxybenzoate, caramel colorant, gardenia colorant, carotene colorant, tar colorant, and the like. In particular embodiments, one or more flavoring agents may be included the contents of the core of the gelatin capsule or in one or more coating layers of the capsule, or a combination thereof. For example, providing a palatable flavoring to the gel capsule may be achieved by providing a flavored coating layer having a water soluble flavor. In such embodiments, from about 0.25 % and about 1.50 % by weight of said coating layer may be the water soluble flavoring. Any suitable flavor known in the art may be provided to the coating layer, such as, berry, strawberry, chocolate, cocoa, vanilla, lemon, nut, almond, cashew, macadamia nut, coconut, blueberry, blackberry, raspberry, peach, lemon, lime, mint, peppermint, orange, banana, chili pepper, pepper, cinnamon, and/or pineapple. In some embodiments, an oil soluble flavoring may be mixed with the active agent that is encapsulated within the capsule. In such embodiments, from about 0.25 % and about 1.50 % by weight of said core may be the oil soluble flavoring. Such oil soluble flavoring may be similar to the taste of the flavor of the capsule, e.g. , strawberry and strawberry, or the taste of the oil flavoring may be complementary to the capsule flavoring, e.g., banana and strawberry. Such flavoring agents and methods for providing flavoring to fatty acid containing capsules may be found in U.S. Patent Nos. 6,346,231 and 6,652,879 which are hereby incorporated by reference in their entireties.

[0086] In some embodiments, the gel capsules of embodiments may include at least one coating layer including one or more secondary agent. In such embodiments, a layer including one or more secondary agent may be of sufficient thickness to prevent oxidative degradation of the one or more secondary agent. For example, in some embodiments, the thickness of this layer may be from about 5 to about 400 microns, about 10 to about 200 microns, about 20 to about 100 microns, or in certain embodiments, from about 40 to about 80 microns. In other embodiments, the thickness of such layers may be expressed in terms of percentage weight gain based on the total weight of the capsule. For example, a layer including one or more secondary agents may create a weight gain of about 0.05 to about 20 %, about 0.1 to about 10%, about 0.1 to about 5%, and in particular embodiments about 0.25 to about 1 %. In certain embodiments, a coating layer containing one or more secondary agent may further include at least one compound to prevent oxidative degradation. For example, in some embodiments, at least one polymer, such as, but not limited to cellulose derivatives such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, polyvinylpyrrolidone/vinyl acetate copolymer, ethyl cellulose aqueous dispersions, and combinations thereof, preferably

hydroxpropyl cellulose, ethyl cellulose, and mixtures thereof, may be added to the coating layer at a ratio of polymer to secondary agent of from about 1 :20 to about 20: 1 by weight or about 1 :5 to about 10: 1 by weight. In particular, where the amount of secondary agent is less than about 15 mg, the amount of polymer may be from about 1 :2 to about 5 : 1 or from about 1 : 1 to about 4: 1, and in embodiments where the amount of secondary agent is about 15 mg or more, the amount of polymer may be from about 1 :4 to about 4: 1 or about 1 :3 to about 2: 1.

[0087] In embodiments in which one or more secondary agents are applied in a coating layer, the secondary agent may be provided as a homogenous coating solution or a heterologous suspension in a pharmaceutically acceptable solvent. Such pharmaceutically acceptable solvents may be an aqueous or organic solvent such as, for example, methanol, ethanol, isopropranol, ethylene glycol, acetone, or mixtures thereof. In other embodiments, pharmaceutically acceptable solvents may include, but are not limited to, polypropylene glycol, polypropylene glycol, polyethylene glycol, for example, polyethylene glycol 600, polyethylene glycol 900, polyethylene glycol 540, polyethylene glycol 1450, polyethylene glycol 6000, polyethylene glycol 8000, and the like; pharmaceutically acceptable alcohols that are liquids at about room temperature, for example, propylene glycol, ethanol, 2-(2-ethoxyethoxy)ethanol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 and the like, polyoxyethylene castor oil derivatives, for example, polyoxyethyleneglycerol triricinoleate or polyoxyl 35 castor oil, polyoxyethyleneglycerol oxystearate, RH 40 (poly ethylenegly col 40 hydrogenated castor oil) or RH 60 (polyethyleneglycol 60 hydrogenated castor oil), and the like, saturated polyglycolized glycerides; polyoxyethylene alkyl ethers, for example, cetomacrogol 1000 and the like; polyoxyethylene stearates, for example, PEG-6 stearate, PEG-8 stearate, polyoxyl 40 stearate NF, polyoxyethyl 50 stearate NF, PEG-12 stearate, PEG-20 stearate, PEG- 100 stearate, PEG-12 distearate, PEG-32 distearate, PEG-150 distearate and the like; ethyl oleate, isopropyl palmitate, isopropyl myristate and the like; dimethyl isosorbide; N- methylpyrrolidinone; parafm; cholesterol; lecithin; suppository bases; pharmaceutically acceptable waxes, for example, carnauba wax, yellow wax, white wax, microcrystalline wax, emulsifying wax and the like; pharmaceutically acceptable silicon fluids; soribitan fatty acid esters such as sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate and the like; pharmaceutically acceptable saturated fats or pharmaceutically acceptable saturated oils, for example, hydrogenated castor oil (glyceryl-tris-12-hydroxystearate), cetyl esters wax (a mixture of primarily C ₁₄ -C ₁₈ saturated esters of C ₁₄ -C ₁₈ saturated fatty acids having a melting range of about 43-47° C), glyceryl monostearate and the like.

[0088] Any method for preparing gel capsules known in the art may be used in various embodiments of the invention. For example, in one embodiment, capsules may be produced by a method including the steps of preparing a sheet of an outer coating layer and one or more sheets of other layers, laminating the sheets, drying the laminated sheets to obtain a dried sheet, and encapsulating the active agent or the compounds and one or more secondary agents within the dried sheet on a rotary filler to form a seamed capsule. In another embodiment, seamless capsules may be produced using an instrument equipped with two or more nozzles arranged concentrically. In other embodiments, gelatin capsules may be manufactured as, for example, a two-piece, sealed or unsealed hard gelatin capsule.

[0089] In another embodiment, a gelatin capsule including the active agent may be formed by the encapsulation of a dose of the active agent in a gelatin capsule. In such embodiments, the gelatin capsule may be made of, for example, gelatin, glycerol, water, a flavoring, a coloring agent and combinations thereof, and the compound dosage may be, for example, 0.001 to 1000 milligrams. One compound dosage range is 0.01 to 500 mg. Preferred compound dosages include 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, and 750 mg of the active ingredient.

[0090] The manufacturing process of such embodiments may include the steps of combining gelswatch ingredients, melting and forming a liquefied gelswatch, delivering the liquefied gelswatch and the active agent to an encapsulation machine, such as the Capsule filler - Zanasi 70C from IMA Pharma (Tonawanda, NY), encapsulating a dose of the compound drying the encapsulated dose, washing the encapsulated dose and packaging the capsules for shipment. The gelswatch ingredients may include any ingredients described herein that are useful in the production of gelatin capsules such as, for example, gelatin or a gelatin substitute such as modified starch or other suitable gelatin substitute known in the art, a softener such as glycerol or sorbitol or other suitable polyol or other gelatin softener known in the art, a flavoring agent such as strawberry flavor Firmenich #52311 A or other suitable gelatin capsule flavoring known in the art and optionally a coloring agent such as keratin or other suitable gelatin capsule coloring agent known in the art.

[0091] In particular embodiments, the gel capsule may be formed from a gelswatch mixture of about 45 parts by weight of gelatin, about 20 parts by weight of glycerol, about 35 parts by weight of water and about 0.5 or more parts by weight of flavoring. The gelswatch ingredients may be heated to about 60° C to 70° C and mixed together to form liquefied gelswatch. The liquefied gelswatch and the active agent may then be poured into an encapsulation machine. The encapsulation machine, such as the Capsule filler - Zanasi 70C from IMA Pharma (Tonawanda, NY), then forms the capsule by encapsulating the dose of the compound into a gelatin capsule.

[0092] The capsule can then be dried at a temperature of, for example, about 20° C. The water content of the capsule may be reduced by evaporation during the drying step. The capsule can then be washed and ready for packaging, selling, or shipping. In some embodiments, a sweetener or flavoring agent can be added to the capsule through a dipping process. In the dipping process, the gelatin capsule is dipped in a sweetener/flavoring solution and then dried, allowing for the sweetener to form a coating around the outside of the capsule. In some embodiments, a sweetener or flavoring agent may be added to the capsule through an enteric coating process, and in other embodiments, a liquefied sweetener or flavoring agent can be sprayed on to the outside of the gelatin capsule and dried. Other methods of making gelatin capsules are known in the art and contemplated.

[0093] In various embodiments, the one or more coatings on the capsule may be applied by any technique known in the art including, but not limited to, pan coating, fluid bed coating or spray coating, and the one or more coatings may be applied, for example, as a solution, suspension, spray, dust or powder. For example, in some embodiments, a polymeric coating may be applied as aqueous-based solutions, organic-based solutions or dispersions containing and, in some embodiments, one or more secondary agent. In such embodiments, polymer-containing droplets may atomized with air or an inert gas and sprayed onto the a core containing the active agent, and in some embodiments, heated air or inert gas may be added to facilitate evaporation of the solvent and film formation. In the case of soft gelatin capsules, the processing parameters of spray rate and bed temperature must be controlled to limit solubilization and capsule

agglomeration. Additionally, a high bed temperature may result in evaporation of residual water from the capsule shell, causing the capsule to become brittle. In addition, coating uniformity which includes mass variance of the coated capsules and variance of the content of the coated active agent and accuracy of deposition must be evaluated.

[0094] Gel capsules of various embodiments of the invention may be of any shape such as, but not limited to, round, oval, tubular, oblong, twist off, or a non-standard shape (e.g., animal, tree, star, heart, etc.), and the size of the capsule may vary in accordance to the volume of the fill composition intended to be contained therein. For example, in some embodiments, hard or soft gelatin capsules may be manufactured using conventional methods as a single body unit comprising the standard capsule shape. A single -body soft gelatin capsule typically may be provided, for example, in sizes from 3 to 22 minims (1 minim = 0.0616 ml) and in shapes of oval, oblong or others. Similarly, hard gel capsules may be manufactured using conventional methods in standard shapes and various standard sizes, such as those designated (000), (00), (0), (1), (2), (3), (4), and (5) where the largest number corresponds to the smallest size. Nonstandard shapes may be used as well.

[0095] Other pharmaceutical formulations containing the compounds of the invention and a suitable carrier can be in various forms including, but not limited to, solids, solutions, powders, fluid emulsions, fluid suspensions, semi-solids, and dry powders including an effective amount of an the active agent of the invention. It is also known in the art that the active ingredients can be contained in such formulations with pharmaceutically acceptable diluents, fillers,

disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, antioxidants, preservatives and the like. The means and methods for administration are known in the art and an artisan can refer to various pharmacologic references for guidance. For example, Modern Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Gilman's, The Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., New York (1980) both of which are hereby incorporated by reference in their entireties can be consulted.

[0096] Other embodiments of the invention include the active agent formulated as a solid dosage form for oral administration including capsules, tablets, pills, powders, and granules. In such embodiments, the active compound may be admixed with one or more inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents and can additionally be prepared with enteric coatings.

[0097] Preparation of an active agent in solid dosage form may vary. For example, in one embodiment, a liquid or gelatin formulation of the active agent may be prepared by combining the active agent with one or more fatty acid diluent, such as those described above, and adding a thickening agent to the liquid mixture to form a gelatin. The gelatin may then be encapsulated in unit dosage form to form a capsule.

[0098] In another exemplary embodiment, an oily preparation or solution of an active agent prepared as described above may be lyophilized or spray dried to form a solid that may be mixed with one or more pharmaceutically acceptable excipient, carrier or diluent to form a tablet, and in yet another embodiment, the active agent may be crystallized to from a solid which may be combined with a pharmaceutically acceptable excipient, carrier or diluent to form a tablet.

[0099] The means and methods for tableting are commonly known in the art and one of ordinary skill in the art can refer to various readily available references for guidance. For example, Pharmaceutical Manufacturing Handbook: Production and Processes, Shayne Cox Gad, John Wiley & Sons, Inc., Hoboken, New Jersey (2008), which is hereby incorporated by reference in its entirety can be consulted.

[00100] Further embodiments which may be useful for oral administration of the active agent include liquid dosage forms. In such embodiments, a liquid dosage may include a

pharmaceutically acceptable emulsion, solution, suspension, syrup, and elixir containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents. Thus, for example, the compounds can 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. Other suitable diluents include, but are not limited to those described below:

[00101] Vegetable oil: As used herein, the term "vegetable oil" refers to a compound, or mixture of compounds, formed from ethoxylation of vegetable oil, wherein at least one chain of polyethylene glycol is covalently bound to the vegetable oil. In some embodiments, the fatty acids may have between about twelve carbons to about eighteen carbons. In some embodiments, the amount of ethoxylation can vary from about 2 to about 200, about 5 to 100, about 10 to about 80, about 20 to about 60, or about 12 to about 18 of ethylene glycol repeat units. The vegetable oil may be hydrogenated or unhydrogenated. Suitable vegetable oils include, but are not limited to castor oil, hydrogenated castor oil, sesame oil, corn oil, peanut oil, olive oil, sunflower oil, safflower oil, soybean oil, benzyl benzoate, sesame oil, cottonseed oil, and palm oil. Other suitable vegetable oils include commercially available synthetic oils such as, but not limited to, Miglyol™ 810 and 812 (available from Dynamit Nobel Chemicals, Sweden) Neobee™ M5 (available from Drew Chemical Corp.), Alofine™ (available from Jarchem Industries), the Lubritab™ series (available from JRS Pharma), the Sterotex™ (available from Abitec Corp.), Softisan™ 154 (available from Sasol), Croduret™ (available from Croda), Fancol™ (available from the Fanning Corp.), Cutina™ HR (available from Cognis), Simulsol™ (available from CJ Petrow), EmCon™ CO (available from Amisol Co.), Lipvol™ CO, SES, and HS-K (available from Lipo), and Sterotex™ HM (available from Abitec Corp.). Other suitable vegetable oils, including sesame, castor, corn, and cottonseed oils, include those listed in R. C. Rowe and P. J. Shesky, Handbook of Pharmaceutical Excipients, (2006), 5th ed., which is incorporated herein by reference in its entirety. Suitable polyethoxylated vegetable oils, include but are not limited to, Cremaphor™ EL or RH series (available from BASF), Emulphor™ EL-719 (available from Stepan products), and Emulphor™ EL-620P (available from GAF).

[00102] Mineral oils: As used herein, the term "mineral oil" refers to both unrefined and refined (light) mineral oil. Suitable mineral oils include, but are not limited to, the Avatech™ grades (available from Avatar Corp.), Drakeol™ grades (available from Penreco), Sirius™ grades (available from Shell), and the Citation™ grades (available from Avater Corp.).

[00103] Castor oils: As used herein, the term "castor oil," refers to a compound formed from the ethoxylation of castor oil, wherein at least one chain of polyethylene glycol is covalently bound to the castor oil. The castor oil may be hydrogenated or unhydrogenated. Synonyms for polyethoxylated castor oil include, but are not limited to polyoxyl castor oil, hydrogenated polyoxyl castor oil, mcrogolglyceroli ricinoleas, macrogolglyceroli hydroxystearas, polyoxyl 35 castor oil, and polyoxyl 40 hydrogenated castor oil. Suitable polyethoxylated castor oils include, but are not limited to, the Nikkol™ HCO series (available from Nikko Chemicals Co. Ltd.), such as Nikkol HCO-30, HC-40, HC-50, and HC-60 (polyethylene glycol-30 hydrogenated castor oil, polyethylene glycol-40 hydrogenated castor oil, polyethylene glycol-50 hydrogenated castor oil, and polyethylene glycol-60 hydrogenated castor oil, Emulphor™ EL-719 (castor oil 40 mole- ethoxylate, available from Stepan Products), the Cremophore™ series (available from BASF), which includes Cremophore RH40, RH60, and EL35 (polyethylene glycol-40 hydrogenated castor oil, polyethylene glycol-60 hydrogenated castor oil, and polyethylene glycol-35 hydrogenated castor oil, respectively), and the Emulgin® RO and HRE series (available from Cognis PharmaLine). Other suitable polyoxy ethylene castor oil derivatives include those listed in R. C. Rowe and P. J. Shesky, Handbook of Pharmaceutical Excipients, (2006), 5th ed., which is incorporated herein by reference in its entirety.

[00104] Sterol: As used herein, the term "sterol" refers to a compound, or mixture of

compounds, derived from the ethoxylation of sterol molecule. Suitable polyethoyxlated sterols include, but are not limited to, PEG-24 cholesterol ether, Solulan™ C-24 (available from

Amerchol); PEG-30 cholestanol, Nikkol™ DHC (available from Nikko); Phytosterol,

GENEROL™ series (available from Henkel); PEG-25 phyto sterol, Nikkol™ BPSH-25

(available from Nikko); PEG-5 soya sterol, Nikkol™ BPS-5 (available from Nikko); PEG- 10 soya sterol, Nikkol™ BPS-10 (available from Nikko); PEG-20 soya sterol, Nikkol™ BPS-20 (available from Nikko); and PEG-30 soya sterol, Nikkol™ BPS-30 (available from Nikko).

[00105] Polyethylene glycol: As used herein, the term "polyethylene glycol" or "PEG" refers to a polymer containing ethylene glycol monomer units of formula -O-CH ₂ -CH ₂ -. Suitable polyethylene glycols may have a free hydroxyl group at each end of the polymer molecule, or may have one or more hydroxyl groups etherified with a lower alkyl, e.g., a methyl group. Also suitable are derivatives of polyethylene glycols having esterifiable carboxy groups. Polyethylene glycols useful in the present invention can be polymers of any chain length or molecular weight, and can include branching. In some embodiments, the average molecular weight of the polyethylene glycol is from about 200 to about 9000. In some embodiments, the average molecular weight of the polyethylene glycol is from about 200 to about 5000. In some embodiments, the average molecular weight of the polyethylene glycol is from about 200 to about 900. In some embodiments, the average molecular weight of the polyethylene glycol is about 400. Suitable polyethylene glycols include, but are not limited to polyethylene glycol-200, polyethylene glycol-300, polyethylene glycol-400, polyethylene glycol-600, and polyethylene glycol-900. The number following the dash in the name refers to the average molecular weight of the polymer. In some embodiments, the polyethylene glycol is polyethylene glycol-400. Suitable polyethylene glycols include, but are not limited to the Carbowax™ and Carbowax™ Sentry series (available from Dow), the Lipoxol™ series (available from Brenntag), the Lutrol™ series (available from BASF), and the Pluriol™ series (available from BASF).

[00106] Propylene glycol fatty acid ester: As used herein, the term "propylene glycol fatty acid ester" refers to an monoether or diester, or mixtures thereof, formed between propylene glycol or polypropylene glycol and a fatty acid. Fatty acids that are useful for deriving propylene glycol fatty alcohol ethers include, but are not limited to, those defined herein. In some embodiments, the monoester or diester is derived from propylene glycol. In some embodiments, the monoester or diester has about 1 to about 200 oxypropylene units. In some embodiments, the

polypropylene glycol portion of the molecule has about 2 to about 100 oxypropylene units. In some embodiments, the monoester or diester has about 4 to about 50 oxypropylene units. In some embodiments, the monoester or diester has about 4 to about 30 oxypropylene units.

Suitable propylene glycol fatty acid esters include, but are not limited to, propylene glycol laurates: Lauroglycol™ FCC and 90 (available from Gattefosse); propylene glycol caprylates: Capryol™ PGMC and 90 (available from Gatefosse); and propylene glycol dicaprylocaprates: Labrafac™ PG (available from Gatefosse).

[00107] Stearoyl macrogol glyceride: Stearoyl macrogol glyceride refers to a polyglycolized glyceride synthesized predominately from stearic acid or from compounds derived

predominately from stearic acid, although other fatty acids or compounds derived from other fatty acids may used in the synthesis as well. Suitable stearoyl macrogol glycerides include, but are not limited to, Gelucire® 50/13 (available from Gattefosse).

[00108] In some embodiments, the diluent component comprises one or more of mannitol, lactose, sucrose, maltodextrin, sorbitol, xylitol, powdered cellulose, microcrystalline cellulose, carboxymethylcellulose, carboxyethylcellulose, methylcellulose, ethylcellulose,

hydroxyethylcellulose, methylhydroxyethylcellulose, starch, sodium starch glycolate, pregelatinized starch, a calcium phosphate, a metal carbonate, a metal oxide, or a metal aluminosilicate.

[00109] Exemplary excipients or carriers for use in solid and/or liquid dosage forms include, but are not limited to:

[00110] Sorbitol: Suitable sorbitols include, but are not limited to, PharmSorbidex E420

(available from Cargill), Liponic 70-NC and 76-NC (available from Lipo Chemical), Neosorb (available from Roquette), Partech SI (available from Merck), and Sorbogem (available from SPI Polyols).

[00111] Starch, sodium starch glycolate, and pregelatinized starch include, but are not limited to, those described in R. C. Rowe and P. J. Shesky, Handbook of Pharmaceutical Excipients,

(2006), 5th ed., which is incorporated herein by reference in its entirety.

[00112] Disintegrant: The disintegrant may include one or more of croscarmellose sodium, carmellose calcium, crospovidone, alginic acid, sodium alginate, potassium alginate, calcium alginate, an ion exchange resin, an effervescent system based on food acids and an alkaline carbonate component, clay, talc, starch, pregelatinized starch, sodium starch glycolate, cellulose floe, carboxymethylcellulose, hydroxypropylcellulose, calcium silicate, a metal carbonate, sodium bicarbonate, calcium citrate, or calcium phosphate.

[00113] Still further embodiments of the invention include the active agent administered in combination with other active such as, for example, adjuvants, anti-inflammatory agents, or other compatible drugs or compounds where such combination is seen to be desirable or advantageous in achieving the desired effects of the methods described herein.

[00114] In another embodiment, the invention includes formulations comprised of liposomes. For example, 5 μΐ of NATOH in acetonitrile (10 mM) was mixed with 4.5 μΐ of Di-lauryl phosphatidyl choline (72.5 mM in chloroform:methanol (1 : 1 v/v). Organic solvents were evaporated by centrifugation under vacuum (Speed Vac) for 1 hr at 30°C. Lipids were resuspended in 1 ml of Tris 20mM, Mes lOmM, NaCl 120mM, DTP A lOOuM and acetic acid lOmM buffer at 50°C for 20 min. Suspension was sonicated at least 10 times at 10% max frequency and till solution became clear. Small Unilamellar Vesicles (SUVs) with NATOH were incubated with b-Mercaptoethanol 250 μΜ from SIGMA (St. Louis, MO). UV-visible spectrums were taken every min up to 10 min. Spectrums were acquired in a Varian Cary 50 Bio from Agilent Technologies (Santa Clara, CA). [00115] In another example, 5 μΐ of NATOH in ACN (10 mM) was mixed with 4.5 μΐ of Di- lauryl phosphatidyl choline (72.5 mM in chloroform:methanol (1 : 1 v/v) and 1.5 μΐ

sphingomyelin (34 mM in chloroform:methanol (1 : 1 v/v). Organic solvents were evaporated by centrifugation under vacuum (Speed Vac) for 1 hr at 30°C. Lipids were resuspended in 1 ml of Tris 20mM, Mes lOmM, NaCl 120mM, DTP A ΙΟΟμΜ and acetic acid lOmM buffer at 50°C for 20 min. Suspension was sonicated at least 10 times at 10% max frequency and till solution became clear. Small Unilamellar Vesicles (SUVs) with NATOH were incubated with b- Mercaptoethanol 250 μΜ from SIGMA (St. Louis, MO). UV -visible spectrums were taken every min up to 10 min. Spectrums were acquired in a Varian Cary 50 Bio from Agilent Technologies (Santa Clara, CA).

[00116] The diameter of liposome vesicles containing the active agent can range from an average diameter from about 50 nm to 70 nm with a preferred average diameter of about 55 nm to about 65 nm, and a more preferred diameter of about 60nm . The average diameter can be measured by light scattering techniques known to one of average skill in the art such as by using a Zetasizer Range by Malvern Instruments Ltd. (Westborough, MA).

[00117] General Synthetic Procedures

[00118] The synthetic route by which the derivatives of a-tocopherol and trolox are obtained is shown in Scheme 1, 2, and 3.

[00119] Scheme 1

[00120] The a-tocopherol is transformed into the corresponding bromoacetate derivative by treatment with bromine followed by O-acetylation. Loss of HBr is avoided and therefore decomposition product formation and dimerization is also avoided. Bromoacetate oxidation with N-methylmorpholine N-oxide yields the corresponding aldehyde. The treatment of this compound with nitromethane in the presence of ammonium acetate gives a product of interest, 5- nitrovinyl-Y-tocopherol (2,7,8-trimethyl-5-((E)-2-nitrovinyl)-2-((4i?,8i?)-4,8, 12- trimethyltridecyl)-3,4-dihydro-2H-chromen-6-ol). Accordingly, treatment with nitroethane, nitropropane, nitrobutane, and so forth in the presence of ammonium acetate results in the longer chain nitroalkenes wherein a π-bond of the nitroalkene remains in conjugation with the chroman ring. The hydroxyl group at position 6 of the chroman ring remains unprotected in the product of interest.

[00121] Scheme 2

[00122] Reactive scheme 2 prepares a trolox derivative with a nitroalkene at position 5 of the chroman ring and an unprotected hydroxyl group at position 6 of the chroman ring.

Accordingly, treatment with nitroethane, nitropropane, nitrobutane, and so forth in the presence of ammonium acetate results in the longer chain nitroalkenes wherein a π-bond of the nitroalkene remains in conjugation with the chroman ring. The hydroxyl group at position 6 of the chroman ring remains unprotected in the product of interest.

[00123] Scheme 3

[00124] Scheme 3 shows a reactive scheme wherein the trolox or tocopherol compound includes longer chain nitroalkenes substituted at position 5 of the chroman ring. The synthesis represented by scheme three contemplates control over the hydrophobicity of the compound through increasing or decreasing the length of the saturated portion of the nitroalkene such that the π-bond is positioned further or closer relative to the chroman ring. For example, the nitroalkene wherein n=l will impart a more hydrophobic character than the nitroalkene wherein n=0 and so on. The scheme above is merely for illustrative purposes. Therefore, although the scheme shows (n=0, 1), the reactive scheme contemplates utilization of longer chain phosporanes in the initial Wittig reaction to dictate the length of the unsaturated portion of the nitroalkene.

[00125] Scheme 4

3 or 6 (R=phytyl or COOCH ₃ ) 12a-b(R=phytyl,COOCH ₃ )

[00126] Instead of nitroalkenes, scheme 4 shows that other electrophilic centers may be introduced at position 5.

[00127] Scheme 5

[00128] In addition to derivatives of tocopherols and trolox, nitroalkenes groups are incorporated at the phytenyl tail of tocotrienols as shown in scheme 5. Scheme 5 includes not only single substitutions of nitro groups on the phytenyl chain but also multiple substitutions of nitro groups by increasing the AgN0 ₂ to tocotrienol mole ratio of the reaction.

[00129] Examples

[00130] The following examples contain detailed methods of preparing compounds of Formula I, Formula II, and Formula III. These detailed descriptions serve to exemplify the above general synthetic schemes which form part of the invention. These detailed descriptions are presented for illustrative purposes only and are not intended as a restriction on the scope of the invention. All parts are by weight and temperatures are in Degrees Celsius unless otherwise indicated. All compounds showed NMR spectra consistent with their assigned structures.

Example 1

2,7,8-trimethyl-5-((E)-2-nitrovm^^

chromen-6-ol (NATOH) [00131] Step 1. Preparation of 5-(bromomethyl)-2,7,8-trimethyl-2-((4R,8R)-4,8, 12- trimethyltridecyl)-3 ,4-dihydro-2H-chromen-6-yl acetate.

[00132] To a solution of a-tocopherol (1, 1.38g, 3.16 mmol) in dry n-hexane (25 mL) a solution of bromine (1.05 equiv.) in n-hexane (10 mL) was added drop-wise at room temperature. The mixture was stirred for 3h. Solvent and remaining bromine were removed in vacuo at room temperature. In the same flask the acetylation reaction was carried out by adding CH2C12 (12 mL), AcOH (12 mL), Ac20 (2.2 mL) and H2S04 (0.2 mL) to obtain the bromomethyl derivative described above. The dark mixture was stirred overnight at room temperature. Then water was added and CH2C12 evaporated. The aqueous phase was extracted with hexane (3x100 mL). The combined organic extracts were subsequently washed to neutrality with water, dried over Na2S04 and concentrated to dryness. Purification by column chromatography (Hex/EtOAc 10: 1) afforded 1.24 g (72% yield) as a yellow dense oil. 1H-NMR, (CDC13/TMS): δ 0.86-0.90 (m, 12H), 1.07-1.59 (m, 24H), 1.77-1.91 (m, 2H, ArCH2CH2), 2.04 (s, 3H, ArCH3), 2.14 (s, 3H, ArCH3), 2.41 (s, 3H, CH3C02), 2.80 (t, J6.6 Hz, 2H, ArCH2CH2).

[00133] Step 2. Preparation of 5-formyl-2,7,8-trimethyl-2-((4R,8R)-4,8,12-trimethyltridecyl )- 3,4-dihydro-2H-chromen-6-yl acetate.

[00134] To the solution of step 1 (1.24 g, 2.24 mmol) in dry acetonitrile (20 mL), NMMO (1.05 g, 9.0 mmol, 4 equiv) was added. After stirring overnight at room temperature, the solvent was evaporated and the crude residue purified by column chromatography (Hex/EtOAc 15:1), affording 3 (1.01 g, 93%) as yellow dense oil. 1H-NMR, (CDC13/TMS): δ 0.86-0.90 (m, 12H), 1.08-1.41 (m, 24H), 1.63-2.09 (m, 2H, ArCH2CH2), 2.09 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3), 2.39 (s, 3H, CH3C02), 3.12 (m, 2H, ArCH2CH2), 10.29(s, 1H).

[00135] Step 3. Preparation of 2,7,8-trimethyl-5-((E)-2-nitrovinyl)-2-((4R,8R)-4,8,12- trimethyltridecyl)-3,4-dihydro-2H-chromen-6-ol (NATOH).

[00136] The solution of step 2 (0.150 g, 0.3 mmol) was added in a mixture of 1.2 mL CH3N02 and equivalent amount of CH3COONH4. The mixture was stirred at 100°C for 2 h. The solvent is then evaporated and H20 and diethyl ether were added. The organic layer was washed with H20 (2x 50 mL), HC1 3N (2x 25 mL), and saturated aqueous NaCl, dried, and the solvent was evaporated. The crude residue purified by column chromatography (Hex/EtOAc 9:1), affording NATOH (0.04 g, 27%) as yellow dense oil. 1H-NMR, (CDC13/TMS): δ 0.85-0.90 (m, 12H), 1.07-1.63 (m, 24H), 1.80-1.92 (m, 2H, ArCH2CH2), 2.21 (s, 6H, ArCH3), 2.88 (t, J2.9Hz, 2H, ArCH2CH2), 8.15(d, J8.2, 2H), 8.32(d, J8.2, 2H). 13C-NMR, (CDC13/TMS): δ 11.54 (2C), 19.23(2C), 20.40(Ar-CH2CH2), 20.71(2C), 22.27 (2C), 23.76(2C), 28.05, 30.59(ArCH2CH2), 31.68(2C), 36.79(4C), 39.29(2C), 74.91, 112.85, 119.37, 120.63, 131.33, 132.73, 139.80, 146.47, 148.08. MS(EI, 70eV):m/z(%): 487(M+, 1), 441(53), 357(55), 287(2).

Example 2

(E)-6-hydroxy-2,7,8-trimethyl-5-(2-nitrovinyl)chromancarb oxylic acid methyl ester

(NA-Tx-ME)

[00137] Step 1. Preparation of 6-hydroxy-2,5,7,8-tetramethylchroman carboxylic acid methyl ester (4).

[00138] Into a 100 mL stainless steel pressure reactor, l,4-dihydroxy-2,3,5-trimethylbenzene (10. Og, 66 mmol), paraformaldehyde (4.00g, 133.2 mmol), methyl methylacrylate (35.0 mL, 327 mmol) and water (3 mL) were placed. The mixture was allowed to react at 180oC for 3 hours, while the reactor was tightly closed. After completion of reaction, the reaction mixture was cooled to room temperature, and methanol was added to the mixture, whereby crystals were precipitated. The crystals were collected through filtration, to thereby yield 6-hydroxy-2, 5,7,8- tetramethylchroman carboxylic acid methyl ester in the form of white powder (11.3 g, 69%). 1H- NMR (CDC13): δ= 1.69 (s, 3H), 1.85-1.93 (m, 1H), 2.18 (s, 3H), 2.21 (m, 3H), 2.43-2.49 (m, 1H), 2.51-2.71 (m, 2H), 3.70 (s, 3H), 4.33 (sa, 1H).

[00139] Step 2. Preparation of 6-acetoxy-5-bromomethyl-2,7,8-trimethylchroman carboxylic acid methyl ester.

[00140] A round-bottom flask was charged with 1.58 g (0.006 mol) of the solution of step 1, 33.0 mL of dichloromethane. The mixture was stirred at room temperature in the dark, and a solution of 0.3 mL (0.006 mol) of bromine in 4.5 mL of dichloromethane was added dropwise. Stirring continued at rt for 2 h after completion of the bromine addition; the resulting solution was dark, but no bromine color or vapor was detectable. The mixture was purged with a stream of nitrogen to remove most of the HBr present, then stripped to dryness under reduce pressure. The crude intermediate product was dissolved with 13 mL of dichloromethane, and treated with 1 1.0 mL of glacial acetic acid, 3.0 mL of acetic anhydride, and 1 drop of cone, sulfuric acid. After stirring overnight at rt, the mixture was treated with 60.0 mL of water and stirred for 1 h. The mixture was transferred to a separatory funnel and the layers were separated. The aqueous layer was extracted with dichloromethane. The combined organic layers were washed with brine, dried (MgS04), and solvent evaporated under reduced pressure. The crude residue purified by column chromatography (Hex/Et20, 7:3), affording 5 (2.0 g, 87% yield) as white solid. 1H- NMR (CDC13): δ 4.48-4.18 (broad, 2H), 3.69 (s, 3H); 2.88-2.81 (m, 1H), 2.68-2.59 (m, 1H), 2.49-2.42(m, 1H), 2.38 (s, 3H), 2.19 (s, 3H), 2.02 (s, 3H), 1.93-1.86 (m, 1H), 1.62 (s, 3H).

[00141] Step 3. Preparation of 6-acetoxy-5-formyl-2,7,8-trimethylchroman carboxylic acid methyl ester.

[00142] A solution of 2g (5.2 mmol) of step 2 in 17 mL of dry acetonitrile treated with 1.58 g (15.6 mmol, 3 eq.) of N-methylmorpholine-N-oxide was stirred at room temperature for 18 h. The mixture was concentrated to about 5mL under reduce pressure. This concentrated solution was poured into water and extracted with ethyl acetate. The combined eorganic layers were washed with 5% HC1 and with brine, dried (MgS04), and solvent evaporated under reduce pressure. The crude residue purified by column chromatography (Hex/Et20, 9: 1), affording 6 (1.2 g, 71% yield) as orange oil that crystallized on stand. 1H-NMR (CDC13): δ 10.22 (s, 1H), 3.71(s, 3H), 3.29-3.22 (m, 1H), 2.92-2.83 (m, 1H), 2.46-2.40 (m, 1H), 2.38(s, 3H), 2.28 (s, 3H), 2.09 (s, 3H), 1.88-1.80 (m, 1H), 1.64(s, 3H).

[00143] Step 4. Preparation (E)-6-hydroxy-2,7,8-trimethyl-5-(2-nitrovinyl)chromancarboxy lic acid methyl ester (NATxME).

[00144] The solution of step 3 (0.18 g, 0.56 mmol) was added in a mixture of 2.5 mL anhyd CH3N02 and equivalent amount of CH3COONH4. The mixture was stirred at 100 oC for 2 h. The solvent is then evaporated and H20 and diethyl ether were added. The organic layer was washed with H20 (2·χ 50 mL), HC1 3 N (2χ·25 mL), and satd aqueous NaCl, dried, and the solvent was evaporated. The crude residue purified by column chromatography (Hex/Et20, 7:3), affording NATxME (0.032 g, 18%) as yellow dense oil. 1H-NMR, (CDC13): δ 1.66 (s, 3H), 1.89-1.98 (m, 1H, ArCH2CH2), 2.20 (s, 3H, ArCH3), 2.28)s, 3H, ArCH3), 2.49-2.54(s, 1H, ArCH2CH2), 2.71-2.79(s, 1H, ArCH2CH2), 2.89-2.97(s, 1H, ArCH2CH2), 8.09(d, J12Hz, 2H), 8.21(d, J12Hz, 2H).

Example 3

[00145] Wittig reaction of aldehyde 3 or 6 with phosphorane 7a or 7b and subsequent acidic hydrolysis gave olefins 8a-d in moderate yields. Then, these derivatives were hydrogenated to produce the corresponding aliphatic aldehydes. The aliphatic aldehydes are added in a mixture of anhydrous CH3N02 and equivalent amounts of CH3COONH4 and stirred at 100 °C for 2 h. The solvent is then evaporated and H20 and diethyl ether are added. The organic layer is washed with H20 (2·χ 50 mL), HC1 3 N (2χ·25 mL), and saturated aqueous NaCl, dried, and the solvent is evaporated. The crude residue is purified by column chromatography (Hex/Et20, 7:3) to afford final products 9a-d in moderate yields. The length of the unsaturated portion of the nitro alkene is dictated by the length of the corresponding unsaturated region in the phosphorane used in the initial Wittig reaction.

Example 4

[00146] Wittig reactions of bromoacetonitrile with aldehydes 3 or 6 in the presence of triphenylphosphine and LiOH in refluxing water containing 1.2 M LiCl for about 20 minutes afford the olefin products 10a or b, respectively, in high yields. Example 5

[00147] Treatment of aldehydes 3 or 6 with ethyl cyanoacetate in the presence of 20 mol % of triphenylphosphine under microwave irradiation at 450W for 5 minutes resulted in the formation of 1 1 a or b with high yields under solvent-free conditions.

Example 6

[00148] Aldehydes 3 and 6 on reaction with carbomethoxy methylene triphenylphosphorane under microwave irradiation (WX-4000, EU Chemicals Instruments) for 2-3 min provided in high yields compounds 12a and 12b, respectively.

Exam le 7

[00149] Alpha-tocotrienol and TEMPO (0.4eq) in combination with AgN02(3 eq) and oven- dried molecular sieves (4 A, 150 mg) in dichloroethane, at 70 °C for 12h gave modest yield of the corresponding nitroalkene derivatives of alpha-tocotrienol. Increasing the AgN0 ₂ to tocotrienol mole ratio resulted in multiple N0 ₂ substitutions on the 3, 7, and 11 positions on the isoprene tail.

[00150] Biologic Activity

[00151] The following general methods are used in order to describe and demonstrate biological activity and potential therapy usage of compounds of the present invention only, and are not to be construed in any way as limiting the scope of the invention.

[00152] In FIG. 1, a Western Blot analysis was performed to analyze HO-1 protein expression after exposure to the NA-Trolox-ME compound. Raw 264.7 cells, a murine macrophage cell line catalog no. TIB-71TM from ATCC (Manassas, VA), were treated with NA-Trolox-ME during 18 hs and after that time cells were lysed. Total protein concentration was measured with Pierce BCA assay from Thermo Fisher Scientific (Rockford, IL). For electrophoresis, 30 ug of protein was used in each line. The proteins were electrophoresed on a Tris/glycine SDS- polyacrylamide gradient gel (10-15%) and transferred to nitrocellulose membrane. The primary antibodies used for detection was rabbit polyclonal anti-HOl from Abeam (Cambridge, MA). Blots were visualized using horseradish peroxidase-conjugated secondary antibodies and ECL Plus detection system from GEHealthcare (Little Chalfont, UK). Blots were detected with a scanner. Protein expression was quantified with ImageQuant TL7.0 software from GE Healthcare (Little Chalfont, UK).

[00153] In FIG. 2, a Western Blot analysis was performed to analyze GCLM protein expression after exposure to the NA-Trolox-ME compound. Raw 264.7 cells, a murine macrophage cell line catalog no. TIB-71TM from ATCC (Manassas, VA), were treated with NA-Trolox-ME for 18 hs were lysed, and total protein concentration was measured with Pierce BCA assay from Thermo Fisher Scientific (Rockford, IL). For electrophoresis, 30 ug of protein was used in each line. The proteins were electrophoresed on a Tris/glycine SDS-polyacrylamide gradient gel (10-15%) and transferred to nitrocellulose membrane. The primary antibodies used for detection was rabbit polyclonal anti-GCLM from Abeam (Cambridge, MA). Blots were visualized using horseradish peroxidase-conjugated secondary antibodies and ECL Plus detection system from GEHealthcare (Little Chalfont, UK). Blots were detected with a scanner. Protein expression was quantified with ImageQuant TL7.0 software from GE Healthcare (Little Chalfont, UK).

[00154] The results shown in FIG. 1 and FIG. 2 illustrate that the nitroalkene tocopherol analog NA-Trolox-ME performs at least similar to electrophilic fatty acid derivatives (positive control OA-NO ₂ ) that mediate cytoprotective cell signaling reactions via phase 2 gene expression.

[00155] In FIG. 3, analysis was performed by Quantitative Reverse Transcription Polymerase Chain Reaction analysis (Q RT-PCR) to detect and quantify mRNA expression levels after exposure to NA-Trolox-ME. Raw 264.7 cells, a murine macrophage cell line catalog no. TIB- 71TM from ATCC (Manassas, VA), were treated with NA-Trolox-ME for 5 hours. RNA extraction was performed using TRIzol ^® from Invitrogen Life Technologies (Carlsbad, CA) reagent according to the manufacturer's instructions. lOOng/ml of RNA was retrotranscripted using the iScript™ cDNA kit from Bio-Rad (Hercules, CA). Samples were analyzed with TaqMan fast universal PCR master mixture using HO-1, GCLM, NQO-1 and GAPDH primers.

[00156] Figure 3 exemplifies the dose dependence of HO-1 protein expression buy the cells.

[00157] In FIGS. 4-6, enzyme-linked immunosorbent assays (ELISA) were performed on raw cells to quantify the concentration of cytokines after exposure to NA-Trolox-ME. Raw 264.7 cells activated with LPS (50 ng/ml) were treated with NA-Trolox-ME. for 18 hours. Cell medium was taken and the concentration of cytokines (TNF-a, MCP-1 & IL-6) was determined using ELISA kits according to the manufacturer's instructions (R & D systems, Minneapolis, MN). [00158] The results shown in FIGS. 4-6 illustrate that the nitroalkene tocopherol analog NA-Tx- TOH is similar to nitro-fatty acid derivatives that potently inhibit stimulus-induced proinflammatory cytokine release from a variety of cells, highlighting the diversity of mechanisms by which nitroalkenes mediate signal transduction. Similar to the endogenous nitroalkenes the NA-Tx-TOH inhibited LPS-induced cytokine release in the macrophage cell line THP-1, with significant reductions observed in interleukin-6 (IL-6), tumor necrosis factor alpha (TNFa), and monocyte chemotractant protein- 1 (MCP-1).

[00159] In FIGS. 7-9, In vivo experiments in mice were performed to detect and quantify mR A transcription levels for HO-1, GCLM, and NQOl after administration of NA-a-TOH. C57BL 6J Mice were treated with NATOH or NA-Trolox-ME (50mg/kg) via intraperitoneal injection or gavage. After 5 hs, mice were sacrificed and organ samples were taken in order to perform Q RT-PCR in vivo analysis of Nrf2/Keapl reporters (HO-1, GCLM & NQOl). Total RNA was extracted with TRIzol ® Invitrogen Life Technologies (Carlsbad, CA) according to

manufacturer's instructions. 100 ng of RNA was retro transcripted using the iScript cDNA kit from Bio-Rad (Hercules, CA). The samples were analyzed by fast TaqMan universal PCR master mixture using HO-1, GCLM, NQO-1 and GAPDH.

[00160] The results in FIGS. 7-9 illustrate the over expression of HO-1, GCLM and NQOl as compared to the positive control groups.

[00161] Further experimental models supporting the present invention include in vitro incorporation of NATOH into low density lipoproteins (LDL). Human and mice LDL purified by sequential ultracentrifugation in KBr gradients was incubated with increasing concentrations of NATOH (-10: 1 molecules per particle ratios). Analysis was performed by reverse phase liquid chromatography mass spectroscopy (RP-LC-MS) of the purified LDL particles after incorporation of the compound.

[00162] Another model includes in vivo incorporation of NATOH into LDL by treating mice with NATOH at a 50mg/kg dose via intraperitoneal injection or gavage. After three days of consecutive administration of the compound, LDL was purified by sequential ultracentrifuation in KBr gradients to show selective secretion of the NATOH into circulation via the VLDL-LDL system by the Alpha-Tocopherol Transfer Protein (a-TTP) after hepatic uptake.

[00163] Other animal models include: (a) atherosclerosis treatment models using rLDL -/- knockout mice and Apo E -/- knockout mice; (b) systemic inflammation or sepsis models using Ccl20 reporter and cecal ligation and perforation; (c) ischemia-reperfusion models characterized by myocardial infarction by isoproterenol and ischemia-reperfusion injury in rat kidneys; and (d) ventilator induced lung injury in rats. The Ccll20 reporter mouse model is a transgenic mouse harboring the transcriptional fusion Ccl20-luciferase as a reporter of pro-inflammatory response. The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin.

[00164] In FIG. 10, NA-a-TOH (100 uM) was incubated with b-Mercaptoethanol (lOmM, SIGMA) in Phosphate buffer 20mM from Sigma- Aldrich (St. Lous, MO) pH = 7.4 with 1 %

SDS. UV-visible spectra was analyzed. Scans were taken each min up to 15 min. Sepectra were acquired in a Varian Cary 50 Bio from Agilent Technologies (Santa Clara, CA).

[00165] The reaction with BME affects this spectrum, decreasing the absorbance at the maximums 420, 350 and 280 nm. During the reaction two isosbestic points (292 & 302 nm) and a new maximum (310 nm) were formed. These characteristic suggest the adduct formation between the nucleophile and the electrophilic nitroalkene-vitamin E analog. The presence of the nitro group on the double bond turns the β-carbon adjacent to the nitroalkene group of the hybrid molecule strongly electrophilic . The hybrid molecule thus reacts covalently with nucleophiles via Michael addition reactions to form an adduct.

[00166] In FIG. 11 the mixture at the end of the reaction described in association with FIG. 10 was analyzed by RP-HPLC (C-18, 2.1 mm ID x 150 mm, 5um, VYDAC from Grace Davison Discovery Sciences (Deerfield, IL)). Column was equilibrated in 50% Acetonitrile from

Mallinckrodt Baker, Inc. (St. Lous, MO) and was eluted by performing a lineal gradient from 50 - 100% of acetonitrile in 10 minutes. Runs were performed using an Agilent 1200 binary HPLC from Agilent Technologies (Santa Clara, CA).

[00167] The figure shows the elution profile of NATOH in a C18 reverse phase column (blue line). After NATOH was incubated with BME, a new more hydrophilic product is formed (green line) that corresponds to the BME-NATOH adduct, as demonstrated by MS experiments (see next slides). Finnally, the reaction with BME does not occur at pH = 2 (red line).

[00168] Chromatographic and mass spectrometric analyses reveal that NATOH is a potent electrophile that can mediate reversible nitroalkylation reactions with both BME and GSH. This occurs by the Michael addition reaction. This reaction also happens with nucleophilic aminoacid residues in proteins (Cys and His) and is viewed to transducer redox- and NO-dependent cell signaling by a covalent, thiol-reversible post-translational modification that regulates protein structure, function, and subcellular distribution.

Treatment

[00169] Compounds of the present invention would be useful for, but not limited to, the treatment of disorders associated with chronic inflammation in a subject, and for treatment of other NRF2/KEAP1 mediated disorders.

[00170] Compounds of the invention would be useful to treat arthritis, including but not limited to rheumatoid arthritis, spondyloarthropathies, gouty arthritis, osteoarthritis, systemic lupus erythematosus and juvenile arthritis. Such compounds of the invention would also be useful in the treatment of asthma, bronchitis, menstrual cramps, preterm labor, tendinitis, bursitis, liver disease including hepatitis, skin related conditions such as psoriasis, eczema, burns and dermatitis, and from post-operative inflammation including from ophthalmic surgery such as cataract surgery and refractive surgery.

[00171] Compounds of the invention also would be useful to treat gastrointestinal conditions such as inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome and ulcerative colitis. Compounds of the invention would be useful in treating inflammation in such diseases as migraine headaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, sclerodoma, rheumatic fever, type I diabetes, neuromuscular junction disease including myasthenia gravis, White matter disease including multiple sclerosis, sarcoidosis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, nephritis, hypersensitivity, swelling occurring after injury including brain edema, myocardial ischemia, and the like.

[00172] The compounds would also be useful in the treatment of ophthalmic diseases, such as retinitis, conjunctivitis, retinopathies, uveitis, ocular photophobia, and of acute injury to the eye tissue.

[00173] The method above would be useful for, but not limited to, treating and preventing inflammation-related cardiovascular disorders in a subject. The method would be useful for treatment and prevention of vascular diseases, coronary artery disease, aneurysm,

arteriosclerosis, atherosclerosis including cardiac transplant atherosclerosis, myocardial inf arction, embolism, stroke, thrombosis, including venous thrombosis, angina including unstable angina, coronary plaque inflammation, bacterial-induced inflammation including Chlamydia- induced inflammation, viral induced inflammation, and inflammation associated with surgical procedures such as vascular grafting including coronary artery bypass surgery, revascularization procedures including angioplasty, stent placement, endarterectomy, or other invasive procedures involving arteries, veins and capillaries.

[00174] The compounds would be useful for, but not limited to, the treatment of angiogenesis- related disorders in a subject. According to the present invention, the compounds are administered to a subject in need of angiogenesis inhibition.

[00175] Treatment may include combination therapies comprising a compound of the present invention with one or more secondary agents including but not limited to the following: statins, beta-blockers, calcium antagonists, angiotensin-converting enzyme inhibitors, inhibitors of angiotensin II receptors, diuretics, and anti-inflammatory agents such as but not limited to acetyl salicylic acid and omega- 3 fatty acids.

[00176] While the invention has been described in connection with various embodiments, it will be understood that the invention is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention, and including such departures from the present disclosure as, within the known and customary practice within the art to which the invention pertains.

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