METHODS OF USE

Cross-Reference

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional

Application Serial No. 62/064,429, filed October 15, 2014, and entitled "FUMARATE

COMPOUNDS, PHARMACEUTICAL COMPOSITIONS, AND METHODS OF USE;" and U.S. Provisional Application Serial No. 62/169,437, filed June 1, 2015, and entitled

"FUMARATE COMPOUNDS, PHARMACEUTICAL COMPOSITIONS, AND METHODS OF USE," the contents of which are incorporated by reference in its entirety.

Field

Disclosed herein are monomethyl and monoethyl fumarates, pharmaceutical

compositions comprising the monomethyl and monoethyl fumarates, and methods of using said monomethyl and monoethyl fumarates and pharmaceutical compositions thereof for treating neurodegenerative, inflammatory, and autoimmune diseases including multiple sclerosis, psoriasis, irritable bowel disorder, ulcerative colitis, arthritis, chronic obstructive pulmonary disease, asthma, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Background

Fumaric acid esters (FAEs) are approved in Germany for the treatment of psoriasis, are being evaluated in the United States for the treatment of psoriasis and multiple sclerosis, and have been proposed for use in treating a wide range of immunological, autoimmune, and inflammatory diseases and conditions.

FAEs and other fumaric acid derivatives have been proposed for use in treating a wide- variety of diseases and conditions involving immunological, autoimmune, and/or inflammatory processes including psoriasis (Joshi and Strebel, WO 1999/49858; US 6,277,882; Mrowietz and Asadullah, Trends Mol Med 2005, 111(1), 43-48; and Yazdi and Mrowietz, Clinics Dermatology 2008, 26, 522-526); asthma and chronic obstructive pulmonary diseases (Joshi et ah, WO 2005/023241 and US 2007/0027076); cardiac insufficiency including left ventricular insufficiency, myocardial infarction and angina pectoris (Joshi et al, WO 2005/023241 ; Joshi et al, US 2007/0027076); mitochondrial and neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, retinopathia pigmentosa and mitochondrial encephalomyopathy (Joshi and Strebel, WO 2002/055063, US 2006/0205659, US 6,509,376, US 6,858,750, and US 7,157,423); transplantation (Joshi and Strebel, WO 2002/055063, US

2006/0205659, US 6,359,003, US 6,509,376, and US 7,157,423; and Lehmann et al, Arch Dermatol Res 2002, 294, 399-404); autoimmune diseases (Joshi and Strebel, WO 2002/055063, US 6,509,376, US 7,157,423, and US 2006/0205659) including multiple sclerosis (MS) (Joshi and Strebel, WO 1998/52549 and US 6,436,992; Went and Lieberburg, US 2008/0089896;

Schimrigk et al., Eur J Neurology 2006, 13, 604-610; and Schilling et al., Clin Experimental Immunology 2006, 145, 101-107); ischemia and reperfusion injury (Joshi et al., US

2007/0027076); advanced glycation end products (AGE)-induced genome damage (Heidland, WO 2005/027899); inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; arthritis; and others ( ilsson et al, WO 2006/037342 and Nilsson and Muller, WO

2007/042034).

The mechanism of action of fumaric acid esters is believed to be mediated by pathways associated with the immunological response. For example, FAEs invoke a shift from a Thl to Th2 immune response, favorably altering the cytokine profile; inhibit cytokine-induced expression of adhesion molecules such as VCAM-1 , ICAM-1 and E-selectin, thereby reducing immune cell extravasation; and deplete lymphocytes through apoptotic mechanisms (Lehmann et al, J Investigative Dermatology 2007, 127, 835-845; Gesser et al, J Investigative Dermatology 2007, 127, 2129-2137; Vandermeeren et al, Biochm Biophys Res Commun 1997, 234, 19-23; and Treumer et al, J Invest Dermatol 2003, 121, 1383-1388).

Recent studies suggest that FAEs are inhibitors of NF-κΒ activation, a transcription factor that regulates the inducible expression of proinflammatory mediators (D'Acquisto et al, Molecular Interventions 2002, 2(1), 22-35). Accordingly, FAEs have been proposed for use in treating NF-κΒ mediated diseases (Joshi et al, WO 2002/055066; and Joshi and Strebel, WO 2002/055063, US 2006/0205659, US 7,157,423 and US 6,509,376). Inhibitors of NF-KB

activation have also been shown to be useful in angiostatic therapy (Tabruyn and Griffioen, Angiogenesis 2008, 11, 101-106), inflammatory bowel disease (Atreya et al, J Intern Med 2008, 263(6), 591-6); and in animal models of diseases involving inflammation including neutrophilic alveolitis, asthma, hepatitis, inflammatory bowel disease, neurodegeneration,

ischemia/reperfusion, septic shock, glomerulonephritis, and rheumatoid arthritis (D'Acquisto et al., Molecular Interventions 2002, 2(1), 22-35).

Studies also suggest that NF-κΒ inhibition by FAEs may be mediated by interaction with tumor necrosis factor (TNF) signaling. Dimethyl fumarate inhibits TNF-induced tissue factor mRNA and protein expression and TNF-induced DNA binding of NF-κΒ proteins, and inhibits the TNF-induced nuclear entry of activated NF-κΒ proteins thereby inhibiting inflammatory gene activation (Loewe et al., J Immunology 2002, 168, 4781-4787). TNF signaling pathways are implicated in the pathogenesis of immune-mediated inflammatory diseases such as rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, juvenile idiopathic arthritis, and ankylosing spondylitis (Tracey et al., Pharmacology & Therapetuics 2008, 117, 244-279).

FUMADERM®, an enteric coated tablet containing a salt mixture of ethyl hydrogen fumarate and dimethyl fumarate (DMF) (2), which is rapidly hydrolyzed to methyl hydrogen fumarate (MHF; also called mono methyl fumarate or MMF) (I) in vivo and is regarded as the main bioactive metabolite, was approved in Germany in 1994 for the treatment of psoriasis.

FUMADERM® is dosed three times/day with 1-2 grams/day administered for the treatment of psoriasis. FUMADERM® exhibits a high degree of interpatient variability with respect to drug absorption and food strongly reduces bioavailability. Absorption is thought to occur in the small intestine with peak levels achieved 5-6 hours after oral administration.

Significant side effects occur in 70-90% of patients (Brewer and Rogers, Clin Expt Ί Dermatology 2007, 32, 246-49; and Hoefnagel et al, Br J Dermatology 2003, 149, 363-369). Side effects of current FAE therapy include gastrointestinal upset including nausea, vomiting, diarrhea, and transient flushing of the skin. Also, DMF exhibits poor aqueous solubility. Fumaric acid derivatives (Joshi and Strebel, WO 2002/055063, US 2006/0205659, and US 7,157,423 (amide compounds and protein- fumarate conjugates); Joshi et al., WO

2002/055066 and Joshi and Strebel, US 6,355,676 (mono and dialkyl esters); Joshi and Strebel, WO 2003/087174 (carbocyclic and oxacarbocylic compounds); Joshi et al., WO 2006/122652 (thiosuccinates); Joshi et al., US 2008/0233185 (dialkyl and diaryl esters)) and salts ( ilsson et al., US 2008/0004344) have been developed in an effort to overcome the deficiencies of current FAE therapy. Controlled release pharmaceutical compositions comprising fumaric acid esters are disclosed by Nilsson and Muller, WO 2007/042034. Glycolamide ester prodrugs are described by Nielsen and Bundgaard, J Pharm Sci 1988, 77(4), 285-298.

Flachsmann et al., US Patent 7,638,118, discloses compounds having the following chemical formula:

wherein:

Z is -OR ² or -Y-(R-NR ³ R ⁴ ) _n ;

R can be a linear or branched C ₂ _9 alkyl;

R ² can be a linear or branched Ci_8 alkyl;

R ³ and R ⁴ , together with the nitrogen atom to which they are bonded, can form an aromatic heterocyclic ring such as a morpholinyl ring; and

when n is 1 , Y can be oxygen.

The compounds are disclosed to be useful for neutralizing odors.

Morpholinoalkyl ester prodrugs of the non-steroidal anti-inflammatory drug niflumic acid exhibit unexpectedly high protection from gastric irritation and ulcerogenicity compared to the parent acid drug (Talath and Gadad, Arzneimittelforschung 2006, 56(11), 744-52). The protective effect is believed to involve absorption of the intact prodrug, which reduces local gastric exposure. Although glycolamide esters of niflumic acid have been synthesized in an effort to improve the biocompatibility of niflumic aid, the effects on gastrointestinal irritation in humans does not appear to have been reported (Talath et al, Arzneimittelforschung 2006, 56(9), 631-9; Gadad et al., Arzneimittelforschung 2002, 52(11), 817-21 ; Benoit et al., Rev. Odontostomatol Midi Fr. 1975, 4, 249-61; and Los et al, Farmaco Sci. 1981 36(5), 372-85). However, the morpholinoalkyl esters, and specifically the morpholinopropyl and morpholinobutyl esters of niflumic acid were identified as exhibiting the best combination of stability, in vivo antiinflammatory activity, and low ulcerogenicity in rats (Talath and Gadad, Arzneimittelforschung 2006, 56(11), 744-52).

Cundy et al., U.S. Patent Publication No. 2013/0203753, discloses morpholinoalkyl fumarates having the followin chemical formula:

or a pharmaceutically acceptable salt thereof, wherein:

n is an integer from 2 to 6;

1 is chosen from methyl, ethyl, C ₃ _ ₆ alkyl, and

m is an integer from 2 to 6.

Zeidan et al., U.S. Patent 8,669,281 discloses compounds having the following general formula:

wherein:

Rl is unsubstituted C1-C6 alkyl;

La is substituted or unsubstituted C1-C6 alkyl linker, substituted or unsubstituted C3-C10 carbocycle, substituted or unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, or substituted or unsubstituted heteroaryl comprising one or two 5- or 6-member rings and 1 -4 heteroatoms selected from , O and S; and R2 and R3 are each, independently, H, substituted or unsubstituted C1 -C6 alkyl, or substituted or unsubstituted C6-C10 aryl; or alternatively, R2 and R3, together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heteroaryl comprising one or two 5- or 6- member rings and 1-4 heteroatoms selected froxm N, O and S or a substituted or unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1 -4 heteroatoms selected from , O and S.

Gangakhedkar et ah, U.S. Patent Publication No. 2010/0048651 , discloses compounds having the following chemical formula:

wherein:

R ¹ and R ² are independently chosen from hydrogen, Ci_ ₆ alkyl, and substituted Ci_ ₆ alkyl;

R ³ and R ⁴ , together with the nitrogen to which they are bonded, can form a C ₅ _io heteroaryl ring such as a morpholino ring; and

R ⁵ can be hydrogen, methyl, ethyl, and C ₃ _ ₆ alkyl;

and pharmaceutical compositions containing such compounds for the treatment of diseases including psoriasis, multiple sclerosis, an inflammatory bowel disease, asthma, chronic obstructive pulmonary disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and arthritis. Compounds in which -NR ³ R ⁴ is a morpholino ring are disclosed in Example 3 (methyl 2-morpholin-4-yl-2-oxoethyl (2E)but-2-ene-l ,4-dioate), Example 28 (methyl l-methyl-2-morpholin-4-yl-2-oxoethyl (2E)but-2-ene-l ,4-dioate), Example 31 ((l S)-l-methyl-2- morpholin-4-yl-2-oxoethyl methyl (2E)but-2-ene- l ,4-dioate), and Example 47 ((2E)-3-[(2- morpholin-4-yl-2-oxoethyl)oxycarbonyl]prop-2-enoic acid).

Summary

Monomethyl and monoethyl fumarate prodrugs having high gastrointestinal permeability and/or absorption, improved solubility, ordered hydrolysis (i.e., preferential cleavage of promoieties), and minimal cleavage in the gut lumen or enterocyte cytoplasm are desirable. Such monomethyl and monoethyl fumarate prodrugs, which provide higher oral bioavailability and plasma levels of the parent monoalkyl fumarate, i.e., monomethyl fumarate or monoethyl fumarate, may enhance the efficacy/responder rate compared to present fumaric acid esters; facilitate the use of lower doses, reduce dosing frequency, and standardize dosing regimens; reduce food effects; reduce gastrointestinal side effects/toxicity; and reduce interpatient treatment variability.

Monomethyl and monoethyl fumarate prodrugs having reduced gastrointestinal side effects are disclosed.

In a first aspect, compounds of Formula (I):

or a pharmaceutically acceptable salt thereof are provided;

wherein:

n is an integer from 1 to 6;

Rl is chosen from methyl and ethyl;

R3 and R4 are each chosen from hydrogen and CI to C6 alkyl; and

R2 is chosen from:

^ ^RS5 and 56 m and p are each 1 or 2;

R5 and RIO are each chosen from hydrogen and methyl;

R6, R7, R8 and R9 are each chosen from hydrogen and a CI to C4 alkyl;

R53 and R54 are each chosen from hydrogen, a halogen, and a CI to C4 alkyl;

L is chosen from -0-, -NH-; -NCH ₃ -, -S-, -S(O)-, and -S(0) ₂ -;

J is chosen from hydrogen, -N0 ₂ ,

X and Y are each chosen from hydrogen, a CI to C4 alkyl, -OCH ₃ , F, CI, and -N0 ₂ ;R55 and R56 are each chosen from hydrogen and CI to C6 alkyl;

with the proviso that when R2 is and R3 and R4 are both hydrogen, then n is not 2.

In certain aspects, L is chosen from -NH-, -NCH ₃ -, -S-, -S(O)-, and -S(0) ₂ -.

In certain aspects, compounds of Formula (I) or a pharmaceutically acceptable salt thereof are provided with the proviso that when R2 is

and R3 and R4 are both hydrogen, then n is not 1. When Rl is methyl, the formula (I) compounds are monomethyl fumarate prodrugs and when Rl is ethyl, the formula (I) compounds are monoethyl fumarate prodrugs.

In a second aspect, compounds of Formula (II):

or a pharmaceutically acceptable salt thereof are provided;

wherein:

Rl 1 is chosen from methyl and ethyl;

R12 is chosen from:

q, r, s and t are each 1 or 2;

R20, R21 , R22 and R23 are each chosen from hydrogen, Ci to C ₄ alkyl, -OCH ₃ , F, CI, and -N0 ₂ ; R13 and R15 are each chosen from hydrogen and -N0 ₂ ;

R14 and R16 are each chosen from hydrogen and methyl;

R17 and R18 are each chosen from hydrogen and CI to C4 alkyl;

R19 is chosen from -0-, -NH-, -NCH ₃ -, -S-, -S(O)-, and -S(0) ₂ -;

R50 and R51 are each chosen from hydrogen, a CI to C4 alkyl,

wherein:

z is 1 or 2; and

R52 is chosen from -0-, -NH-; -NCH ₃ -, -S-, -S(O)-, and -S(0) ₂ -.

When Rl 1 is methyl, the formula (II) compounds are monomethyl fumarate prodrugs and when Rl 1 is ethyl, the formula (II) compounds are monoethyl fumarate prodrugs.

In a third aspect, compounds of Formula (III):

or a pharmaceutically acceptable salt thereof are provided;

wherein:

R24 is chosen from methyl and ethyl;

R25 and R26 are each chosen from hydrogen and CI to C4 alkyl;

R27 is chosen from:

R28 is chosen from hydrogen, methyl, ethyl and benzyl;

R29 is chosen from methyl, ethyl and benzyl;

R30 is chosen from hydrogen and methyl;

R31 is chosen from a Ci to C ₄ alkyl, benzyl and aryl;

R32 is chosen from a CI to C4 alkyl, benzyl and CI to C4 alkyl substituted with benzyl;

R33 and R39 are each chosen from hydrogen and -N0 ₂ ;

R34 and R40 are each chosen from hydrogen and methyl;

R35 is chosen from hydrogen, a Ci to C ₄ alkyl, -OCH ₃ , F, CI, and -N0 ₂ ;

R36 and R37 are each chosen from hydrogen and a CI to C4 alkyl;

R38 is chosen from hydrogen, methyl and ethyl;

R41 and R42 are each chosen from hydrogen and a CI to C4 alkyl;

R43 is chosen from -OH and -NH ₂ ;

R44 is chosen from -0-, -NH-; -NCH ₃ -, -S-, -S(O)-, and -S(0) ₂ -; M is chosen from -C-, -0-, -N-, and -S-;

u is an integer from 1 to 6; and

v, w, x, and y are each 1 or 2.

In certain aspects, R44 is chosen from -NH-, -NCH ₃ -, -S-, -S(O)-, and -S(0) ₂ -;

When R24 is methyl, the formula (III) compounds are monomethyl fumarate prodrugs and when R24 is ethyl, the formula (III) compounds are monoethyl fumarate prodrugs.

In a fourth aspect, compounds of Formula (IV):

or a pharmaceutically acceptable salt thereof are provided;

wherein:

R46 is chosen from methyl and ethyl; and

R47 and R48 are each chosen from hydrogen and a CI to C4 alkyl.

When R46 is methyl, the formula (IV) compounds are monomethyl fumarate prodrugs and when R46 is ethyl, the formula (IV) compounds are monoethyl fumarate prodrugs.

In a fifth aspect, pharmaceutical compositions comprising a pharmaceutically acceptable vehicle and a therapeutically effective amount of a compound of formula (I), (II), (III), or (IV) are provided.

Such compounds and pharmaceutical compositions are useful for treating

neurodegenerative, inflammatory and autoimmune diseases and disorders including, for example, multiple sclerosis, psoriasis, irritable bowel disorder, ulcerative colitis, arthritis, chronic obstructive pulmonary disease, asthma, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.

In another aspect, methods of treating a disease in a patient are provided comprising administering to a patient in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (I), (II), (III), or (IV). In certain embodiments, the disease is chosen from a neurodegenerative disease, an inflammatory disease, and an autoimmune disease including, for example, multiple sclerosis, psoriasis, irritable bowel disorder, ulcerative colitis, arthritis, chronic obstructive pulmonary disease, asthma, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Detailed Description

Definitions

A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a moiety or substituent. For example, -CONH ₂ is bonded through the carbon atom.

"Alkyl" refers to a saturated or unsaturated, branched, or straight-chain, monovalent hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene, or alkyne. Examples of alkyl groups include, for example, methyl; ethyls such as ethanyl, ethenyl, and ethynyl; propyls such as propan-l-yl, propan-2-yl, prop-l-en-l-yl, prop-l-en-2-yl, prop-2-en-l-yl (allyl), prop- 1 -yn- 1 -yl, prop-2-yn- 1 -yl, etc.; butyls such as butan- 1 -yl, butan-2-yl, 2-methyl-propan- 1 -yl, 2-methyl-propan-2-yl, but- 1 -en- 1 -yl,

but-l-en-2-yl, 2-methyl-prop-l-en-l-yl, but-2-en- 1 -yl, but-2-en-2-yl, buta-l,3-dien-l-yl, buta-l,3-dien-2-yl, but- 1 -yn- 1 -yl, but-l-yn-3-yl, but-3-yn-l-yl, etc., and the like.

The term "alkyl" includes groups having any degree or level of saturation, i.e., groups having exclusively single carbon-carbon bonds, groups having one or more double

carbon-carbon bonds, groups having one or more triple carbon-carbon bonds, and groups having combinations of single, double, and triple carbon-carbon bonds. Where a specific level of saturation is intended, the terms alkanyl, alkenyl, or alkynyl are used. In certain embodiments, an alkyl group can have from 1 to 10 carbon atoms (C _1-10 ), in certain embodiments, from 1 to 6 carbon atoms (Ci_ ₆ ), in certain embodiments from 1 to 4 carbon atoms (C _1-4 ), in certain embodiments, from 1 to 3 carbon atoms (C _1-3 ), and in certain embodiments, from 1 to 2 carbon atoms (C _1-2 ). In certain embodiments, alkyl is methyl, in certain embodiments, ethyl, and in certain embodiments, n-propyl or isopropyl.

"Compounds" of Formulae (I), (II), (III), and (IV) disclosed herein include any specific compounds within this formula. Compounds may be identified either by their chemical structure and/or chemical name. Compounds are named using Chemistry 4-D Draw Pro, version 7.01c (Chemlnnovation Software, Inc., San Diego, CA). When the chemical structure and chemical name conflict, the chemical structure is determinative of the identity of the compound. The compounds described herein may comprise one or more chiral centers and/or double bonds and therefore may exist as stereoisomers such as double -bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. Accordingly, any chemical structures within the scope of the specification depicted, in whole or in part, with a relative configuration encompass all possible enantiomers and stereoisomers of the illustrated compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures may be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art. Compounds of Formulae (I), (II), (III), and (IV) include, for example, optical isomers of compounds of Formulae (I), (II), (III), and (IV), racemates thereof, and other mixtures thereof. In such embodiments, a single enantiomer or diastereomer, i.e., optically active form can be obtained by asymmetric synthesis or by resolution of the racemates. Resolution of the racemates may be accomplished, for example, by methods such as crystallization in the presence of a resolving agent, or

chromatography using, for example, chiral stationary phases. Notwithstanding the foregoing, in compounds of Formulae (I), (II), (III), and (IV) the configuration of the illustrated double bond is only in the E configuration (i.e., trans configuration).

Compounds of Formulae (I), (II), (III), and (IV) also include isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature. Examples of isotopes that may be incorporated into the compounds disclosed herein include, for example, ² H, ³ H, ⁿ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ 0, ¹⁷ 0, etc.

Compounds may exist in unsolvated forms as well as solvated forms, including hydrated forms and as N-oxides. In general, compounds disclosed herein may be free acid, hydrated, solvated, or N-oxides. Certain compounds may exist in multiple crystalline, co-crystalline, or amorphous forms. Compounds of Formulae (I), (II), (III), and (IV) include pharmaceutically acceptable salts thereof or pharmaceutically acceptable solvates of the free acid form of any of the foregoing, as well as crystalline forms of any of the foregoing. Compounds of Formulae (I), (II), (III), and (IV) also include solvates. A solvate refers to a molecular complex of a compound with one or more solvent molecules in a stoichiometric or non-stoichiometric amount. Such solvent molecules include those commonly used in the pharmaceutical art, which are known to be innocuous to a patient, e.g., water, ethanol, and the like. A molecular complex of a compound or moiety of a compound and a solvent can be stabilized by non-covalent intra-molecular forces such as, for example, electrostatic forces, van der Waals forces, or hydrogen bonds. The term "hydrate" refers to a solvate in which the one or more solvent molecules are water.

Further, when partial structures of the compounds are illustrated, an asterisk (*) indicates the point of attachment of the partial structure to the rest of the molecule.

"Disease" refers to a disease, disorder, condition, or symptom of any of the foregoing.

"Drug" as defined under 21 U.S.C. § 321(g)(1) means "(A) articles recognized in the official United States Pharmacopoeia, official Homeopathic Pharmacopoeia of the United States, or official National Formulary, or any supplement to any of them; and (B) articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or other animals; and (C) articles (other than food) intended to affect the structure or any function of the body of man or other animals . . ."

"Leaving group" has the meaning conventionally associated with it in synthetic organic chemistry, i.e., an atom or a group capable of being displaced by a nucleophile and includes halogen such as chloro, bromo, fluoro, and iodo; acyloxy, such as acetoxy and benzoyloxy, alkoxycarbonylaryloxycarbonyl, mesyloxy, tosyloxy, and trifluoromethanesulfonyloxy; aryloxy such as 2,4-dinitrophenoxy, methoxy, N,0-dimethylhydroxylamino, /?-nitrophenolate, imidazolyl, and the like.

The terms "MHF", "MMF", "monomethyl fumarate" and "methyl hydrogen fumarate" are synonymous, all referring to a compound having the following chemical structure:

The terms "EHF", "MEF", "monoethyl fumarate" and "ethyl hydrogen fumarate" are synonymous, all referring to a compound having the following chemical structure:

"Parent heteroaromatic ring system" refers to an aromatic ring system in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom in such a way as to maintain the continuous π-electron system characteristic of aromatic systems and a number of out-of-plane π-electrons corresponding to the Huckel rule {An +2). Examples of heteroatoms to replace the carbon atoms include, for example, N, P, O, S, and Si, etc. Specifically included within the definition of "parent heteroaromatic ring systems" are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, arsindole, benzodioxan, benzofuran, chromane, chromene, indole, indoline, xanthene, etc. Examples of parent heteroaromatic ring systems include, for example, arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, thiazolidine, oxazolidine, and the like.

"Patient" refers to a mammal, for example, a human.

"Pharmaceutically acceptable" refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

"Pharmaceutically acceptable salt" refers to a salt of a compound that possesses the desired pharmacological activity of the parent compound. Such salts include acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-l -carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; and salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine,

N-methylglucamine, and the like. In certain embodiments, a pharmaceutically acceptable salt is the hydrochloride salt. In certain embodiments, a pharmaceutically acceptable salt is the sodium salt.

"Pharmaceutically acceptable vehicle" refers to a pharmaceutically acceptable diluent, a pharmaceutically acceptable adjuvant, a pharmaceutically acceptable excipient, a

pharmaceutically acceptable carrier, or a combination of any of the foregoing with which a compound provided by the present disclosure may be administered to a patient, which does not destroy the pharmacological activity thereof and which is non-toxic when administered in doses sufficient to provide a therapeutically effective amount of the compound or a pharmacologically active metabolite thereof.

"Pharmaceutical composition" refers to a compound of Formulae (I), (II), (III), and (IV), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable vehicle, with which the compound of Formulae (I), (II), (III), and (IV), or a pharmaceutically acceptable salt thereof, is administered to a patient.

"Treating" or "treatment" of any disease refers to reversing, alleviating, arresting, or ameliorating a disease or at least one of the clinical symptoms of a disease, reducing the risk of acquiring a disease or at least one of the clinical symptoms of a disease, inhibiting the progress of a disease or at least one of the clinical symptoms of the disease or reducing the risk of developing a disease or at least one of the clinical symptoms of a disease. "Treating" or

"treatment" also refers to inhibiting a disease, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both, and to inhibiting at least one physical parameter that may or may not be discernible to the patient. In certain embodiments, "treating" or "treatment" refers to delaying the onset of a disease or at least one or more symptoms thereof in a patient who may be exposed to or predisposed to a disease even though that patient does not yet experience or display symptoms of the disease.

"Therapeutically effective amount" refers to the amount of a compound that, when administered to a subject for treating a disease, or at least one of the clinical symptoms of a disease, is sufficient to effect such treatment of the disease or symptom thereof. The

"therapeutically effective amount" may vary depending, for example, on the compound, the disease and/or symptoms of the disease, severity of the disease and/or symptoms of the disease, the age, weight, and/or health of the patient to be treated, and the judgment of the prescribing physician. An appropriate amount in any given compound may be ascertained by those skilled in the art and/or is capable of determination by routine experimentation.

"Therapeutically effective dose" refers to a dose that provides effective treatment of a disease in a patient. A therapeutically effective dose may vary from compound to compound and/or from patient to patient, and may depend upon factors such as the condition of the patient and the route of delivery. A therapeutically effective dose may be determined in accordance with routine pharmacological procedures known to those skilled in the art.

Reference is now made in detail to certain embodiments of compounds, compositions, and methods. The disclosed embodiments are not intended to be limiting of the claims. To the contrary, the claims are intended to cover all alternatives, modifications, and equivalents.

Compounds

Certain embodiments provide a compound of Formula (I):

or a pharmaceutically acceptable salt thereof;

wherein:

n is an integer from 1 to 6; Rl is chosen from methyl and ethyl;

R3 and R4 are each chosen from hydrogen and CI to C6 alkyl; and

R2 is chosen from:

^ ^Rs5

I

and ^R 56 m and p are each 1 or 2;

R5 and RIO are each chosen from hydrogen and methyl;

R6, R7, R8 and R9 are each chosen from hydrogen and a CI to C4 alkyl;

R53 and R54 are each chosen from hydrogen, a halogen, and a CI to C4 alkyl;

L is chosen from -0-, -NH-; -NCH ₃ -, -S-, -S(O)-, and -S(0) ₂ -;

J is chosen from hydrogen, -N0 ₂ ,

X and Y are each chosen from hydrogen, a CI to C4 alkyl, -OCH ₃ , F, CI, and -N0 ₂ ; R55 and R56 are each chosen from hydrogen and CI to C6 alkyl;

with the proviso that when R2 is and R3 and R4 are both hydrogen, then n is not 2.

Alternatively, compounds of Formula (I) or a pharmaceutically acceptable salt thereof are provided, wherein:

n is an integer from 1 to 6;

Rl is chosen from methyl and ethyl;

R3 and R4 are each chosen from hydrogen and CI to C6 alkyl; and

R2 is chosen from:

m and p are each 1 or 2;

R5 and RIO are each chosen from hydrogen and methyl;

R6, R7, R8 and R9 are each chosen from hydrogen and a CI to C4 alkyl;

R53 and R54 are each chosen from hydrogen, a halogen, and a CI to C4 alkyl;

L is chosen from -NH-, -NCH ₃ -, -S-, -S(O)-, and -S(0) ₂ -;

J is chosen from hydrogen, -N0 ₂ ,

X and Υ are each chosen from hydrogen, a CI to C4 alkyl, -OCH ₃ , F, CI, and -N0 ₂ ;

R55 and R56 are each chosen from hydrogen and CI to C6 alkyl;

with the proviso that when R2 is and R3 and R4 are both hydrogen, then n is not 2; and

with the proviso that when R2 is

and R3 and R4 are both hydrogen, then n is not 1.

Examples of Formula (I) compounds include:

Methyl (2-oxopyrrolidinyl)methyl (2E)but- dioate,

Methyl 2-(2-oxopyrrolidinyl)ethyl (2E)but-2-ene-l ,4- dioate, Methyl 3-(2-oxopyrrolidinyl)propyl (2E)but-2-ene-

1 ,4-dioate,

Methyl 2-pyrrolidinylethyl (2E) but-2-ene-l,4-dioate,

Methyl 2-pyrrolidinylbutyl (2E) but-2-ene-l,4- dioate,

l-{2-[(2E)-3-(methoxycarbonyl)prop-2- enoyloxy]ethyl}pyrrolidine-2-carboxylic acid,

l-{2-[(2E)-3-(methoxycarbonyl)prop-2- enoyloxy]butyl}pyrrolidine-2-carboxylic acid, and pharmaceutically acceptable salts thereof.

Additional examples of Formula (I) compounds include: dimethylamino)ethyl methyl (2E)but-2-ene-l,4-dioate, 3-(dimethylamino)propyl methyl (2E)but-2-ene-l ,4- dioate,

4-(dimethylamino)butyl methyl (2E)but-2-ene- -dioate,

5-(dimethylamino)pentyl methyl (2E)but-2- ene-l,4-dioate,

6-(dimethylamino)hexyl methyl (2E)but-2- ene-l,4-dioate,

2-(diethylamino)ethyl methyl (2E)but-2-ene- 1 ,4- dioate, (diethylamino)propyl methyl (2E)but-2-ene-l,4-

4-(diethylamino)butyl methyl (2E)but-2-ene-

1 ,4-dioate,

5-(diethylamino)pentyl methyl (2E)but-2- ene-l,4-dioate,

6-(diethylamino)hexyl methyl (2E)but- 2-ene-l ,4-dioate, and pharmaceutically acceptable salts thereof.

Yet additional examples of Formula (I) compound include:

Methyl 2-piperazinylethyl (2E)but-2-ene-l,4-dioate,

Methyl 3-piperazinylpropyl (2E)but-2-ene-l,4- dioate, and pharmaceutically acceptable salts thereof.

Certain embodiments provide a compound of Formula (II): 2

or a pharmaceutically acceptable salt thereof;

wherein:

Rl 1 is chosen from methyl and ethyl;

R12 is chosen from:

q, r, s and t are each 1 or 2;

R20, R21 , R22 and R23 are each chosen from hydrogen, Ci to C ₄ alkyl, -OCH ₃ , F, CI, and -NO

R13 and R15 are each chosen from hydrogen and -N0 ₂ ;

R14 and R16 are each chosen from hydrogen and methyl;

R17 and R18 are each chosen from hydrogen and CI to C4 alkyl;

R19 is chosen from -0-, -NH-, -NCH ₃ -, -S-, -S(0)-, and -S(0) ₂ -;

R50 and R51 are each chosen from hydrogen, a CI to C4 alkyl,

wherein:

z is 1 or 2; and

R52 is chosen from -0-, -NH-; -NCH ₃ -, -S-, -S(O)-, and -S(0) ₂ - Examples of compounds of Formula (II) include:

l phenyl (2E)but-2-ene-l,4-dioate,

-chlorophenyl methyl (2E)but-2-ene- 1 ,4-dioate,

ethyl 4-methylphenyl (2E)but-2-ene- 1 ,4-dioate,

Methyl 4-nitrophenyl (2E)but-2-ene-l,4-dioate, 2,6-bis(methylethyl)phenyl methyl (2E)but-2-ene-l ,4-dioate,

Cyclohexyl methyl (2E)but-2-ene-l,4-dioate,

Cyclopentyl methyl (2E)but-2-ene-l,4-dioate, 2H-3,4,5,6-tetrahydropyran-2-yl methyl (2E)but-2-ene-l,4- dioate,

methylamino methyl (2E)but-2-ene-l,4-dioate, Diethylamino methyl (2E)but-2-ene- 1 ,4-dioate, Methyl piped dyl (2E)but-2-ene- 1 ,4-dioate, and pharmaceutically acceptable salts thereof.

Certain embodiments provide a compound of Formula (III):

or a pharmaceutically acceptable salt thereof;

wherein:

R24 is chosen from methyl and ethyl;

R25 and R26 are each chosen from hydrogen and CI to C4 alkyl;

R27 is chosen from:

R28 is chosen from hydrogen, methyl, ethyl, and benzyl;

R29 is chosen from methyl, ethyl, and benzyl;

R30 is chosen from hydrogen and methyl;

R31 is chosen from a Ci to C ₄ alkyl, benzyl, and aryl;

R32 is chosen from a CI to C4 alkyl, benzyl and CI to C4 alkyl substituted with benzyl;

R33 and R39 are each chosen from hydrogen and -N0 ₂ ;

R34 and R40 are each chosen from hydrogen and methyl;

R35 is chosen from hydrogen, a Ci to C ₄ alkyl, -OCH ₃ , F, CI, and -N0 ₂ ;

R36 and R37 are each chosen from hydrogen and a CI to C4 alkyl;

R38 is chosen from hydrogen, methyl, and ethyl;

R41 and R42 are each chosen from hydrogen and a CI to C4 alkyl;

R43 is chosen from -OH and -NH ₂ ;

R44 is chosen from -0-, -NH-, -NCH ₃ -, -S-, -S(O)-, and -S(0) ₂ -;

M is chosen from -C-, -0-, -N-, and -S-;

u is an integer from 1 to 6; and

v, w, x, and y are each 1 or 2.

In certain aspects, R44 is chosen from -NH-, -NCH ₃ -, -S-, -S(O)-, and -S(0) ₂ -. Examples of compounds of Formula (III) include:

(Methoxycarbonyl)methyl methyl (2E)but-2-ene-l ,4- dioate, (ethoxycarbonyl)methyl methyl (2E)but-2-ene- 1 ,4- dioate, Methyl [benzyloxycarbonyljmethyl (2E)but-2- ene-l,4-dioate,

Methyl oxolan-2-ylmethyl (2E)but-2-ene-l ,4-dioate, thyl (2-oxopyrrolidinyl) (2E)but-2-ene-l ,4-dioate, 5-Dioxoazolidinyl)methyl (2E)but-2-ene-l,4-dioate, (Methoxy-N-methylcarbonylamino)methyl methyl (2E)but-2-ene- 1 ,4-dioate, (Ethoxy-N-methylcarbonylamino)methyl methyl

(2E)but-2-ene- 1 ,4-dioate,

Methyl [N- methyl(phenylmethoxy)carbonylamino]methyl (2E)but-2-ene-l,4-dioate, and pharmaceutically acceptable salts thereof.

Certain embodiments provide a compound of Formula (IV):

or a pharmaceutically acceptable salt thereof;

wherein:

R46 is chosen from methyl and ethyl; and

R47 and R48 are each chosen from hydrogen and a CI to C4

Examples of compounds of Formula (IV) include:

2-{(2E)-3-(methoxycarbonyl)prop-2-enoylamino]propanoic acid and pharmaceutically acceptable salts thereof.

Pharmaceutical Compositions

Pharmaceutical compositions provided by the present disclosure may comprise a therapeutically effective amount of a compound of Formulae (I), (II), (III), or (IV) together with a suitable amount of one or more pharmaceutically acceptable vehicles so as to provide a composition for proper administration to a patient. Suitable pharmaceutical vehicles are described in the art.

In certain embodiments, a compound of Formulae (I), (II), (III), or (IV) may be incorporated into pharmaceutical compositions to be administered orally. Oral administration of such pharmaceutical compositions may result in uptake of a compound of Formulae (I), (II), (III), or (IV) throughout the intestine and entry of such compound into the systemic circulation. Such oral compositions may be prepared in a manner known in the pharmaceutical art and comprise a compound of Formulae (I), (II), (III), and (IV) and at least one pharmaceutically acceptable vehicle. Oral pharmaceutical compositions may include a therapeutically effective amount of a compound of Formulae (I), (II), (III), and (IV) and a suitable amount of a pharmaceutically acceptable vehicle, so as to provide an appropriate form for oral administration to a patient.

Compounds of Formula Formulae (I), (II), (III), and (IV) may be incorporated into pharmaceutical compositions to be administered by any appropriate route of administration including intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical. In certain embodiments, compounds of Formulae (I), (II), (III), and (IV)may be incorporated into pharmaceutical compositions to be administered orally.

Pharmaceutical compositions comprising a compound of Formulae (I), (II), (III), and (IV) and may be manufactured by means of conventional mixing, dissolving, granulating,

dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries, which facilitate processing of compounds of Formulae (I), (II), (III), and (IV), or crystalline forms thereof and one or more pharmaceutically acceptable vehicles into formulations that can be used

pharmaceutically. Proper formulation is in part dependent upon the route of administration chosen. Pharmaceutical compositions provided by the present disclosure may take the form of solutions, suspensions, emulsions, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for administration to a patient.

Pharmaceutical compositions provided by the present disclosure may be formulated in a unit dosage form. A unit dosage form refers to a physically discrete unit suitable as a unitary dose for patients undergoing treatment, with each unit containing a predetermined quantity of a compound of Formulae (I), (II), (III), and (IV) calculated to produce an intended therapeutic effect. A unit dosage form may be for a single daily dose, for administration 2 times per day, or one of multiple daily doses, e.g., 3 or more times per day. When multiple daily doses are used, a unit dosage form may be the same or different for each dose. One or more dosage forms may comprise a dose, which may be administered to a patient at a single point in time or during a time interval.

Pharmaceutical compositions comprising a compound of Formulae (I), (II), (III), and (IV) may be formulated for immediate release.

In certain embodiments, an oral dosage form provided by the present disclosure may be a controlled release dosage form. Controlled delivery technologies can improve the absorption of a drug in a particular region, or regions, of the gastrointestinal tract. Controlled drug delivery systems may be designed to deliver a drug in such a way that the drug level is maintained within a therapeutically effective window and effective and safe blood levels are maintained for a period as long as the system continues to deliver the drug with a particular release profile in the gastrointestinal tract. Controlled drug delivery may produce substantially constant blood levels of a drug over a period of time as compared to fluctuations observed with immediate release dosage forms. For some drugs, maintaining a constant blood and tissue concentration of the drug throughout the course of therapy is the most desirable mode of treatment. Immediate release of drugs may cause blood levels to peak above a level required to elicit a desired response, which may waste the drug and/or may cause or exacerbate toxic side effects. Controlled drug delivery can result in optimum therapy, and not only can reduce the frequency of dosing, but may also reduce the severity of side effects. Examples of controlled release dosage forms include dissolution controlled systems, diffusion controlled systems, ion exchange resins, osmotically controlled systems, erodable matrix systems, pH independent formulations, and gastric retention systems. An appropriate oral dosage form for a particular pharmaceutical composition provided by the present disclosure may depend, at least in part, on the gastrointestinal absorption properties of a compound of Formulae (I), (II), (III), and (IV), the stability of a compound of Formulae (I), (II), (III), and (IV) in the gastrointestinal tract, the pharmacokinetics of a compound of Formulae (I), (II), (III), and (IV) and the intended therapeutic profile. An appropriate controlled release oral dosage form may be selected for a particular compound of Formulae (I), (II), (III), and (IV). For example, gastric retention oral dosage forms may be appropriate for compounds absorbed primarily from the upper gastrointestinal tract, and sustained release oral dosage forms may be appropriate for compounds absorbed primarily from the lower gastrointestinal tract. Certain compounds are absorbed primarily from the small intestine. In general, compounds traverse the length of the small intestine in about 3 to 5 hours. For compounds that are not easily absorbed by the small intestine or that do not dissolve readily, the window for active agent absorption in the small intestine may be too short to provide a desired therapeutic effect.

In certain embodiments, pharmaceutical compositions provided by the present disclosure may be practiced with dosage forms adapted to provide sustained release of a compound of

Formulae (I), (II), (III), and (IV) upon oral administration. Sustained release oral dosage forms may be used to release drugs over a prolonged time period and are useful when it is desired that a drug or drug form be delivered to the lower gastrointestinal tract. Sustained release oral dosage forms include any oral dosage form that maintains therapeutic concentrations of a drug in a biological fluid such as the plasma, blood, cerebrospinal fluid, or in a tissue or organ for a prolonged time period. Sustained release oral dosage forms include diffusion-controlled systems such as reservoir devices and matrix devices, dissolution-controlled systems, osmotic systems, and erosion-controlled systems. Sustained release oral dosage forms and methods of preparing the same are well known in the art.

An appropriate dose of a compound of Formulae (I), (II), (III), and (IV), or

pharmaceutical composition comprising a compound of Formulae (I), (II), (III), and (IV), may be determined according to any one of several well-established protocols. For example, animal studies, such as studies using mice, rats, dogs, and/or monkeys, may be used to determine an appropriate dose of a pharmaceutical compound. Results from animal studies may be extrapolated to determine appropriate doses for use in other species, such as for example, humans.

Uses

Compounds of Formulae (I), (II), (III), and (IV) are derivatives of monoalkyl hydrogen fumarates. Thus, compounds of Formulae (I), (II), (III), and (IV) and pharmaceutical compositions thereof may be administered to a patient suffering from any disease including a disorder, condition, or symptom for which monoalkyl hydrogen fumarates are known or hereafter discovered to be therapeutically effective. Indications for which mo no methyl fumarate (MMF) has been prescribed, and hence for which a compound of Formulae (I), (II), (III), and (IV), or pharmaceutical compositions thereof are also expected to be effective, include psoriasis. Other indications for which compounds of Formulae (I), (II), (III), and (IV) may be

therapeutically effective include multiple sclerosis, irritable bowel disorder, ulcerative colitis, arthritis, chronic obstructive pulmonary disease, asthma, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.

Methods of treating a disease in a patient provided by the present disclosure comprise administering to a patient in need of such treatment a therapeutically effective amount or dose of a compound of Formulae (I), (II), (III), and (IV). Compounds of Formulae (I), (II), (III), and (IV) or pharmaceutical compositions thereof may provide therapeutic or prophylactic plasma and/or blood concentrations of fumarate following administration to a patient.

Compounds of Formulae (I), (II), (III), and (IV) may be included in a pharmaceutical composition and/or dosage form adapted for oral administration, although compounds of Formulae (I), (II), (III), and (IV) may also be administered by any other appropriate route, such as for example, by injection, infusion, inhalation, transdermally, or absorption through epithelial or mucosal membranes (e.g., oral, rectal, and/or intestinal mucosa).

Compounds of Formulae (I), (II), (III), and (IV) may be administered in an amount and using a dosing schedule as appropriate for treatment of a particular disease. Daily doses of compounds of Formulae (I), (II), (III), and (IV) may range from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 50 mg/kg, from about 1 mg/kg to about 50 mg/kg, and in certain embodiments, from about 5 mg/kg to about 25 mg/kg. In certain embodiments, compounds of Formulae (I), (II), (III), and (IV) may be administered at a dose over time from about 1 mg to about 5 g per day, from about 10 mg to about 4 g per day, and in certain embodiments from about 20 mg to about 2 g per day. An appropriate dose of a compound of Formulae (I), (II), (III), and (IV) may be determined based on several factors, including, for example, the body weight and/or condition of the patient being treated, the severity of the disease being treated, the incidence and/or severity of side effects, the manner of administration, and the judgment of the prescribing physician. Appropriate dose ranges may be determined by methods known to those skilled in the art.

Compounds of Formulae (I), (II), (III), and (IV) may be assayed in vitro and in vivo for the desired therapeutic or prophylactic activity prior to use in humans. In vivo assays, for example using appropriate animal models, may also be used to determine whether administration of a compound of Formulae (I), (II), (III), and (IV) is therapeutically effective.

In certain embodiments, a therapeutically effective dose of a compound of Formulae (I),

(II) , (III), and (IV) may provide therapeutic benefit without causing substantial toxicity including adverse side effects. Toxicity of compounds of Formulae (I), (II), (III), and (IV) and/or metabolites thereof may be determined using standard pharmaceutical procedures and may be ascertained by those skilled in the art. The dose ratio between toxic and therapeutic effect is the therapeutic index. A dose of a compound of Formulae (I), (II), (III), and (IV) may be within a range capable of establishing and maintaining a therapeutically effective circulating plasma and/or blood concentration of a compound of Formulae (I), (II), (III), and (IV) that exhibits little or no toxicity.

Compounds of Formulae (I), (II), (III), and (IV) may be used to treat diseases, disorders, conditions, and symptoms of any of the foregoing for which alkyl hydrogen fumarates, such as MMF, are known to provide or are later found to provide therapeutic benefit. MMF is known to be effective in treating psoriasis, multiple sclerosis, an inflammatory bowel disease, asthma, chronic obstructive pulmonary disease, and arthritis. Hence, compounds of Formulae (I), (II),

(III) , and (IV) may also be used to treat any of these diseases and disorders.

Compounds of Formulae (I), (II), (III), and (IV) or pharmaceutical salts thereof may be used to treat any of the following diseases and disorders: immunological, autoimmune, and/or inflammatory diseases including psoriasis, arthritis, asthma, and chronic obstructive pulmonary disease; cardiac insufficiency including left ventricular insufficiency, myocardial infarction, and angina pectoris; mitochondrial and neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, retinopathia pigmentosa, and mitochondrial encephalomyopathy; transplantation rejection; autoimmune diseases such as multiple sclerosis; ischemia and reperfusion injury; AGE-induced genome damage; inflammatory bowel diseases such as Crohn's disease, irritable bowel disorder, and ulcerative colitis; and NF-κΒ mediated diseases; COPD, rheumatica, granuloma annulare, lupus, autoimmune carditis, eczema, sarcoidosis, and autoimmune diseases including acute

disseminated encephalomyelitis, Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, Bechet's disease, celiac disease, Chagas disease, chronic obstructive pulmonary disease, Crohn's disease, dermatomyositis, diabetes mellitus type I, endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hidradenitis suppurativea, Kawasaki disease, IgA neuropathy, idiopathic thrombocytopenic purpura, interstitial cystitis, lupus erythematosus, mixed connective tissue disease, morphea, multiple sclerosis, myasthenia gravis, narcolepsy, neuro myotonia, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, schizophrenia, scleroderma, Sjogren's syndrome, stiff person syndrome, temporal arteritis, ulcerative colitis, vasculitis, vitiligo, acute disseminated encephalomyelitis, myasthenia gravis, Wegener's granulomatosis, adrenal leukodystrophy, Alexanders disease, Alper's Disease, balo concentric sclerosis, bronchiolitis obliterans organizing pneumonia, Canavan disease, central nervous system vasculitic syndrome, Charcot- Marie-Tooth disease, childhood ataxia with central nervous system hypomyelination, chronic inflammatory demyelinating polyneuropathy (CIDP), cutaneous lupus erythematosus (CLE), chronic lymphocytic leukemia (CLL), diabetic retinopathy, globoid cell leukodystrophy, graft versus host disease (GVHD), hepatitis C (HCV), herpes simplex viral infection, human immunodeficiency virus (HIV), lichen planus, macular degeneration, monomelic amyotrophy, necrobiosis lipoidosis, neurodegeneration with brain iron accumulation, neuromyelitis optica, neurosarcoidosis, optic neuritis, a pareneoplastic syndrome, Pelizaeus-Merzbacher disease, Primary lateral sclerosis, progressive supranuclear palsy, Schilder's Disease, subacute necrotizing myelopathy ₃ Susac's syndrome, transverse myelitis, a tumor, and Zellweger spectrum.

The underlying etiology of any of the foregoing diseases being treated may have a multiplicity of origins. Further, in certain embodiments, a therapeutically effective amount of one or more compounds of Formulae (I), (II), (III), and (IV) may be administered to a patient, such as a human, as a preventative measure against various diseases or disorders. Thus, a therapeutically effective amount of one or more compounds of Formulae (I), (II), (III), and (IV) may be administered as a preventative measure to a patient having a predisposition for and/or history of immunological, autoimmune, and/or inflammatory diseases including psoriasis, arthritis, asthma, and chronic obstructive pulmonary disease; cardiac insufficiency including left ventricular insufficiency, myocardial infarction, and angina pectoris; mitochondrial and neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, retinopathia pigmentosa, and mitochondrial

encephalomyopathy; transplantation rejection; autoimmune diseases such as multiple sclerosis; ischemia and reperfusion injury; AGE-induced genome damage; inflammatory bowel diseases such as Crohn's disease, irritable bowel disorder, and ulcerative colitis; and F-κΒ mediated diseases.

Psoriasis

Psoriasis is characterized by hyperkeratosis and thickening of the epidermis as well as by increased vascularity and infiltration of inflammatory cells in the dermis. Psoriasis vulgaris manifests as silvery, scaly, erythematous plaques on typically the scalp, elbows, knees, and buttocks. Guttate psoriasis occurs as tear-drop size lesions.

Fumaric acid esters are recognized for the treatment of psoriasis and dimethyl fumarate is approved for the systemic treatment of psoriasis in Germany (Mrowietz and Asadullah, Trends Mol Med 2005, 11(1), 43-48; and Mrowietz et al, Br J Dermatology 1999, 141, 424-429).

Efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating psoriasis can be determined using animal models and in clinical trials.

Inflammatory Arthritis

Inflammatory arthritis includes diseases such as rheumatoid arthritis, juvenile rheumatoid arthritis (juvenile idiopathic arthritis), psoriatic arthritis, and ankylosing spondylitis produce joint inflammation. The pathogenesis of immune-mediated inflammatory diseases including inflammatory arthritis is believed to involve TNF and NF-κΒ signaling pathways (Tracey et al., Pharmacology & Therapeutics 2008, 117, 244-279). DMF has been shown to inhibit TNF and inflammatory diseases including inflammatory arthritis, which are believed to involve TNF and NK-KB signaling, and therefore may be useful in treating inflammatory arthritis (Lowewe et al., J Immunology 2002, 168, 4781-4787).

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating inflammatory arthritis can be determined using animal models and in clinical trials.

Multiple Sclerosis

Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system caused by an autoimmune attack against the insulating axonal myelin sheaths of the central nervous system. Demyelination leads to the breakdown of conduction and to severe disease with destruction of local axons and irreversible neuronal cell death. The symptoms of MS are highly varied with each individual patient exhibiting a particular pattern of motor, sensible, and sensory disturbances. MS is typified pathologically by multiple inflammatory foci, plaques of demyelination, gliosis, and axonal pathology within the brain and spinal cord, all of which contribute to the clinical manifestations of neurological disability {see e.g., Wingerchuk, Lab Invest 2001, 81, 263-281 ; and Virley, NeuroRx 2005, 2(4), 638-649). Although the causal events that precipitate MS are not fully understood, evidence implicates an autoimmune etiology together with environmental factors, as well as specific genetic predispositions. Functional impairment, disability, and handicap are expressed as paralysis, sensory and octintive

disturbances, spasticity, tremor, a lack of coordination, and visual impairment, which impact the quality of life of the individual. The clinical course of MS can vary from individual to individual, but invariability of the disease can be categorized in three forms: relapsing-remitting, secondary progressive, and primary progressive.

Studies support the efficacy of fumaric acid esters for treating MS, which are presently undergoing phase II clinical testing (Schimrigk et al., Eur J Neurology 2006, 13, 604-610; and Wakkee and Thio, Current Opinion Investigational Drugs 2007, 8(11), 955-962).

Assessment of MS treatment efficacy in clinical trials can be accomplished using tools such as the Expanded Disability Status Scale and the MS Functional as well as magnetic resonance imaging lesion load, biomarkers, and self-reported quality of life. Animal models of MS shown to be useful to identify and validate potential therapeutics include experimental autoimmune/allergic encephalomyelitis (EAE) rodent models that simulate the clinical and pathological manifestations of MS and nonhuman primate EAE models.

Inflammatory Bowel Disease ( Crohn 's Disease, Ulcerative Colitis)

Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the large intestine and in some cases, the small intestine that includes Crohn's disease and ulcerative colitis. Crohn's disease, which is characterized by areas of inflammation with areas of normal lining in between, can affect any part of the gastrointestinal tract from the mouth to the anus. The main gastrointestinal symptoms are abdominal pain, diarrhea, constipation, vomiting, weight loss, and/or weight gain. Crohn's disease can also cause skin rashes, arthritis, and inflammation of the eye. Ulcerative colitis is characterized by ulcers or open sores in the large intestine or colon. The main symptom of ulcerative colitis is typically constant diarrhea with mixed blood of gradual onset. Other types of intestinal bowel disease include collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Bechet's colitis, and indeterminate colitis.

FAEs are inhibitors of NF-κΒ activation and therefore may be useful in treating inflammatory diseases such as Crohn's disease and ulcerative colitis (Atreya et al., J Intern Med 2008, 263(6), 59106).

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating inflammatory bowel disease can be evaluated using animal models and in clinical trials. Useful animal models of inflammatory bowel disease are known.

Irritable Bowel Syndrome

Irritable bowel syndrome is a disorder that affects the large intestine and is typically characterized by abdominal pain or cramping, a bloated feeling, flatulence, diarrhea or constipation and/or mucus in the stool.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating irritable bowel syndrome can be evaluated using animal models and in clinical trials. Useful animal models of inflammatory bowel disease are known. Asthma

Asthma is reversible airway obstruction in which the airway occasionally constricts, becomes inflamed, and is lined with an excessive amount of mucus. Symptoms of asthma include dyspnea, wheezing, chest tightness, and cough. Asthma episodes may be induced by airborne allergens, food allergies, medications, inhaled irritants, physical exercise, respiratory infection, psychological stress, hormonal changes, cold weather, or by other factors.

As an inhibitor of NF-κΒ activation and as shown in animal studies (Joshi et al., US 2007/0027076) FAEs may be useful in treating pulmonary diseases such as asthma and chronic obstructive pulmonary disorder.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating asthma can be assessed using animal models and in clinical trials.

Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD), also known as chronic obstructive airway disease, is a group of diseases characterized by the pathological limitation of airflow in the airway that is not fully reversible, and includes conditions such as chronic bronchitis, emphysema, as well as other lung disorders such as asbestosis, pneumoconiosis, and pulmonary neoplasms (see, e.g., Barnes, Pharmacological Reviews 2004, 56(4), 515-548). The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases. COPD is characterized by a shortness of breath that lasts for months or years, possibly accompanied by wheezing, and a persistent cough with sputum production. COPD is most often caused by tobacco smoking, although it can also be caused by other airborne irritants such as coal dust, asbestos, urban pollution, or solvents. COPD encompasses chronic obstructive bronchiolitis with fibrosis and obstruction of small airways, and emphysema with enlargement of airspaces and destruction of lung parenchyma, loss of lung elasticity, and closure of small airways.

The efficacy of administering at least one compound of Formulae (I), (II), (III), and (IV) for treating chronic obstructive pulmonary disease may be assessed using animal models of chronic obstructive pulmonary disease and in clinical studies. For example, murine models of chronic obstructive pulmonary disease are known. Neurodegenerative Disorders

Neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease,

Huntington's disease and amyotrophic lateral sclerosis are characterized by progressive dysfunction and neuronal death. NF-κΒ inhibition has been proposed as a therapeutic target for neurodegenerative diseases (Camandola and Mattson, Expert Opin Ther Targets 2007, 11(2), 123-32).

Parkinson 's Disease

Parkinson's disease is a slowly progressive degenerative disorder of the nervous system characterized by tremor when muscles are at rest (resting tremor), slowness of voluntary movements, and increased muscle tone (rigidity). In Parkinson's disease, nerve cells in the basal ganglia, e.g., substantia nigra, degenerate, and thereby reduce the production of dopamine and the number of connections between nerve cells in the basal ganglia. As a result, the basal ganglia are unable to control smooth muscle movements and coordinate changes in posture as normal, leading to tremor, incoordination, and slowed, reduced movement (bradykinesia) (Blandini, et al, Mol. Neurobiol. 1996, 12, 73-94).

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating Parkinson's disease may be assessed using animal and human models of Parkinson's disease and in clinical studies.

Alzheimer's Disease

Alzheimer's disease is a progressive loss of mental function characterized by

degeneration of brain tissue, including loss of nerve cells and the development of senile plaques and neurofibrillary tangles. In Alzheimer's disease, parts of the brain degenerate, destroying nerve cells and reducing the responsiveness of the maintaining neurons to neurotransmitters. Abnormalities in brain tissue consist of senile or neuritic plaques, e.g., clumps of dead nerve cells containing an abnormal, insoluble protein called amyloid, and neurofibrillary tangles, twisted strands of insoluble proteins in the nerve cell.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating Alzheimer's disease may be assessed using animal and human models of Alzheimer's disease and in clinical studies. Huntington 's Disease

Huntington's disease is an autosomal dominant neurodegenerative disorder in which specific cell death occurs in the neostriatum and cortex (Martin, N Engl J Med 1999, 340, 1970- 80). Onset usually occurs during the fourth or fifth decade of life, with a mean survival at age of onset of 14 to 20 years. Huntington's disease is universally fatal, and there is no effective treatment. Symptoms include a characteristic movement disorder (Huntington's chorea), cognitive dysfunction, and psychiatric symptoms. The disease is caused by a mutation encoding an abnormal expansion of CAG-encoded polyglutamine repeats in the protein, huntingtin.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating Huntington's disease may be assessed using animal and human models of Huntington's disease and in clinical studies.

Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the progressive and specific loss of motor neurons in the brain, brain stem, and spinal cord (Rowland and Schneider, N Engl J Med 2001, 344, 1688-1700). ALS begins with weakness, often in the hands and less frequently in the feet that generally progresses up an arm or leg. Over time, weakness increases and spasticity develops characterized by muscle twitching and tightening, followed by muscle spasms and possibly tremors. The average age of onset is 55 years, and the average life expectancy after the clinical onset is 4 years. The only recognized treatment for ALS is riluzole, which can extend survival by only about three months.

The efficacy compounds of Formulae (I), (II), (III), and (IV) for treating ALS may be assessed using animal and human models of ALS and in clinical studies.

Adrenal Leukodystrophy

Adrenal leukodystrophy (which is also sometimes referred to as adrenoleukodystrophy) describes several closely related inherited disorders that disrupt the breakdown (metabolism) of certain fats (very-long-chain fatty acids). Adrenal leukodystrophy is passed down from parents to their children as an X-linked genetic trait. It therefore affects mostly males, although some women who are carriers can have milder forms of the disease. It affects approximately 1 in 20,000 people from all races. The condition results in the buildup of very-long-chain fatty acids in the nervous system, adrenal gland, and testes, which disrupts normal activity. The disease is closely related to Schilder's disease, marked by diffuse abnormality of the cerebral white matter and adrenal atrophy. The disease is characterized by mental deterioration progressing to dementia, and by aphasia, apraxia, dysarthria, and loss of vision in about a third of the patients. Almost all patients show abnormal adrenal functioning when tested.Currently, adrenal leukodystrophy is treated with steroids such as Cortisol, eating a diet low in very-long-chain fatty acids and taking Lorenzo's oil, which can lower the blood levels of very-long-chain fatty acids. Bone marrow transplants are also being tested as an experimental treatment.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating adrenal leukodystrophy can be determined using animal models and in clinical trials. Suitable animal models for adrenal leukodystrophy are disclosed for example in Fourcade, S., et al. (2010) Hum Mol Genet 19(10), 2005-14; and Khan, M., et al. (2008) J Neurochem 106(4), 1766-79.

Alexanders Disease

Alexanders disease (which is also sometimes referred to as Alexander disease) is one of a group of neurological conditions known as the leukodystrophies, disorders that are the result of abnormalities in myelin, the "white matter" that protects nerve fibers in the brain. Alexanders disease is a progressive and usually fatal disease. The destruction of white matter is accompanied by the formation of Rosenthal fibers, which are abnormal clumps of protein that accumulate in non-neuronal cells of the brain called astrocytes. Rosenthal fibers are sometimes found in other disorders, but not in the same amount or area of the brain that are featured in Alexanders disease. The infantile form is the most common type of Alexanders disease. It has an onset during the first two years of life. Usually there are both mental and physical developmental delays, followed by the loss of developmental milestones, an abnormal increase in head size, and seizures. The juvenile form of Alexanders disease is less common and has an onset between the ages of two and thirteen. These children may have excessive vomiting, difficulty swallowing and speaking, poor coordination, and loss of motor control. Adult-onset forms of Alexanders disease are rare, but have been reported. The symptoms sometimes mimic those of Parkinson's disease or multiple sclerosis. The disease occurs in both males and females, and there are no ethnic, racial, geographic, or cultural economic differences in its distribution.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating Alexanders Disease can be determined using animal models and in clinical trials. Suitable animal models for Alexanders Disease are disclosed for example in Wang, L., et al. (2011) J Neurosci 31(8), 2868- 77.

Alpers ' Disease

Alpers' disease (which is also referred to as Christensen-Krabbe disease, poliodystrophia cerebri, and progressive cerebral or progressive infantile poliodystrophy) is a rare disease of young children, characterized by neuronal degeneration of the cerebral cortex and elsewhere, accompanied by progressive mental deterioration, motor disturbances, seizures, and early death. The disease is a progressive, neurodevelopmental, mitochondrial DNA depletion syndrome characterized by three co-occurring clinical symptoms: psychomotor regression (dementia); seizures; and liver disease. It is an autosomal recessive disease caused by mutation in the gene for the mitochondrial DNA polymerase POLG. The disease occurs in about one in 100,000 persons. Most individuals with Alpers' disease do not show symptoms at birth and develop normally for weeks to years before the onset of symptoms. Diagnosis is established by testing for the POLG gene. Symptoms typically occur months before tissue samples show the mitochondrial DNA depletion, so that these depletion studies cannot be used for early diagnosis. About 80 percent of individuals with Alpers' disease develop symptoms in the first two years of life, and 20 percent develop symptoms between ages 2 and 25. The first symptoms of the disorder are usually nonspecific and may include hypoglycemia secondary to underlying liver disease, failure to thrive, infection-associated encephalopathy, spasticity, myoclonus (involuntary jerking of a muscle or group of muscles), seizures, or liver failure. An increased protein level is seen in cerebrospinal fluid analysis. Cortical blindness (loss of vision due to damage to the area of the cortex that controls vision) develops in about 25 percent of cases. Gastrointestinal dysfunction and cardiomyopathy may occur. Dementia is typically episodic and often associated with an infection that occurs while another disease is in process. Seizures may be difficult to control and unrelenting seizures can cause developmental regression as well. "Alpers-like" disorders without liver disease are genetically different and have a different clinical course. Fewer than one-third of individuals with the "Alpers-like" phenotype without liver disease have POLG mutations.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating Alper's Disease can be determined using animal models and in clinical trials. Balo Concentric Sclerosis

Balo concentric sclerosis (also sometimes referred to as Balo's disease, encephalitis periaxialis concentrica, leukoencephalitis periaxialis concentrica, and concentric sclerosis) is an atypical form of Schilder's disease in which the demyelination is arranged in concentric rings around a central circle.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating balo concentric sclerosis can be determined using animal models and in clinical trials.

Bronchiolitis Obliterans Organizing Pneumonia

Bronchiolitis obliterans organizing pneumonia (also sometimes referred to as BOOP and/or cryptogenic organizing pneumonia) is a non-infectious pneumonia; specifically, an inflammation of the bronchioles (bronchiolitis) and surrounding tissue in the lungs. It is often a complication of an existing chronic inflammatory disease such as rheumatoid arthritis, or it can be a side effect of certain medications such as amiodarone.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating bronchiolitis obliterans organizing pneumonia can be determined using animal models and in clinical trials. Suitable animal models for bronchiolitis obliterans organizing pneumonia are disclosed for example in Majeski et al., Respiratory reovirus 1/L induction of intraluminal fibrosis, a model of bronchiolitis obliterans organizing pneumonia, is dependent on T lymphocytes, Am J Pathol. (2003 Oct), 163(4): 1467-79; and Gillen et al., Rapamycin blocks fibrocyte migration and attenuates bronchiolitis obliterans in a murine model Ann Thorac Surg. (2013 May), 95(5): 1768- 75.

Canavan disease

Canavan disease, one of the most common cerebral degenerative diseases of infancy, is a gene-linked, neurological birth disorder in which the brain degenerates into spongy tissue riddled with microscopic fluid-filled spaces. Canavan disease has been classified as one of a group of genetic disorders known as the leukodystrophies but, unlike most leukodystrophies, both grey and white matter are severely affected in infants with Canavan disease. Recent research has indicated that the cells in the brain responsible for making myelin sheaths, known as

oligodendrocytes, cannot properly complete this critical developmental task. Myelin sheaths are the fatty covering that act as insulators around nerve fibers in the brain, as well as providing nutritional support for nerve cells. In Canavan disease, many oligodendrocytes do not mature and instead die, leaving nerve cell projections known as axons vulnerable and unable to properly function. Canavan disease is caused by mutation in the gene for an enzyme called aspartoacylase, which acts to break down the concentrated brain chemical known as N-acetyl-aspartate.

Symptoms of Canavan disease usually appear in the first 3 to 6 months of life and progress rapidly. Symptoms include lack of motor development, feeding difficulties, abnormal muscle tone (weakness or stiffness), and an abnormally large, poorly controlled head. Paralysis, blindness, or hearing loss may also occur. Children are characteristically quiet and apathetic. Although Canavan disease may occur in any ethnic group, it is more frequent among Ashkenazi Jews from eastern Poland, Lithuania, and western Russia, and among Saudi Arabians. Canavan disease can be identified by a simple prenatal blood test that screens for the missing enzyme or for mutations in the gene that controls aspartoacylase. Both parents must be carriers of the defective gene in order to have an affected child. When both parents are found to carry the Canavan gene mutation, there is a one in four (25 percent) chance with each pregnancy that the child will be affected with Canavan disease.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating Canavan disease can be determined using animal models and in clinical trials. Suitable animal models for Canavan disease are disclosed for example in Madhavarao, C. N., et al. (2009) J Inherit Metab Dis 32(5), 640-50.

Central Nervous System Vasculitis

Central nervous system vasculitis is an inflammation of, in and around blood vessels, which includes the veins, arteries, and capillaries, and secondary narrowing or blockage of the blood vessels that nourish the brain and spinal cord. Researchers think that inflammation occurs with infection or is thought to be due to a faulty immune system response.

A central nervous system vasculitic syndrome may begin suddenly or develop over time.

Symptoms include: headaches, especially a headache that doesn't go away; fever; feeling out-of- sorts; rapid weight loss; confusion or forgetfulness leading to dementia; aches and pains in the joints and muscles; pain while chewing or swallowing; paralysis or numbness, usually in the arms or legs; and visual disturbances, such as double vision, blurred vision, or blindness. The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating central nervous system vasculitis can be determined using animal models and in clinical trials. Suitable animal models for central nervous system vasculitis are disclosed for example in Malipiero, U., et al. (2006) Brain 129(9), 2404-15.

Charcott-Marie-Tooth Disease

Charcot-Marie-Tooth disease is a muscular atrophy of variable inheritance, beginning in the muscles supplied by the peroneal nerves and progressing slowly to involve the muscles of the hands and arms. The disease is also called Charcot-Marie atrophy or syndrome, peroneal or peroneal muscular atrophy, Marie-Tooth disease, and Tooth's disease. Charcot-Marie-Tooth disease is one of the most common inherited neurological disorders, affecting approximately 1 in 2,500 people in the United States. The disease is named for the three physicians who first identified it in 1886 - Jean-Martin Charcot and Pierre Marie in Paris, France, and Howard Henry Tooth in Cambridge, England. Charcot-Marie-Tooth disease, also known as hereditary motor and sensory neuropathy or peroneal muscular atrophy, comprises a group of disorders that affect peripheral nerves. The peripheral nerves lie outside the brain and spinal cord and supply the muscles and sensory organs in the limbs. Disorders that affect the peripheral nerves are called peripheral neuropathies.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating Charcott- Marie-Tooth Disease can be determined using animal models and in clinical trials. Suitable animal models for Charcott-Marie-Tooth Disease are disclosed for example in Fledrich, R., et al. (2012) Br Med Bull 102(1), 89-113.

Childhood Ataxia with Central Nervous System Hypomyelination

Childhood ataxia with central nervous system hypomyelination is characterized by ataxia, spasticity, and variable optic atrophy. The phenotypic range includes a prenatal/congenital form, a subacute infantile form (onset age <1 year), an early childhood onset form (onset age 1-5 years), a late childhood /juvenile onset form (onset age 5-15 years), and an adult onset form. The prenatal/congenital form is characterized by severe encephalopathy. In the later onset forms initial motor and intellectual development is normal or mildly delayed followed by neurologic deterioration with a chronic progressive or subacute course. Chronic progressive decline can be exacerbated by rapid deterioration during febrile illnesses or following head trauma or major surgical procedures, or by acute psychological stresses such as extreme fright.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating childhood ataxia with central nervous system hypomyelination can be determined using animal models and in clinical trials. Suitable animal models for childhood ataxia with central nervous system hypomyelination are disclosed for example in Geva, M., et al. (2010) Brain 133(8), 2448-61. Chronic Inflammatory Demyelinating Polyneuropathy

Chronic inflammatory demyelinating polyneuropathy (CIDP) is a neurological disorder characterized by progressive weakness and impaired sensory function in the legs and arms. The disorder, which is sometimes called chronic relapsing polyneuropathy, is caused by damage to the myelin sheath (the fatty covering that wraps around and protects nerve fibers) of the peripheral nerves. Although it can occur at any age and in both genders, CIDP is more common in young adults, and in men more so than women. It often presents with symptoms that include tingling or numbness (beginning in the toes and fingers), weakness of the arms and legs, loss of deep tendon reflexes (areflexia), fatigue, and abnormal sensations. CIDP is closely related to Guillain-Barre syndrome and it is considered the chronic counterpart of that acute disease.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating CIDP can be determined using animal models and in clinical trials. Suitable animal models for CIDP are disclosed for example in Ubogu E.E., et al. (2012) Journal of the Peripheral Nervous System 17(1), 53-61 ; Sajic M. et al. (2012) PLoS ONE 7(2):Article Number: e30708; and Meyer Zu Horste G, et al. (2011) Journal of the Peripheral Nervous System 16(SUPPL. 3), S87.

Cutaneous Lupus Erythematosus

Cutaneous lupus erythematosus (CLE) can be divided into three main subtypes: acute, subacute, and chronic, all of which demonstrate photosensitivity. Acute cutaneous lupus erythematosus (ACLE) most commonly presents as symmetric erythema overlying the malar cheeks and nasal bridge with sparing of the nasolabial folds (butterfly rash). However, it can also present as a diffuse morbilliform eruption with erythema and edema of the hands, with prominent sparing of the joints. Subacute cutaneous lupus erythematosus (SCLE)

characteristically presents as annular or psoriasiform plaques in a photodistribution. Chronic cutaneous lupus erythematosus (CCLE) can be further divided into 3 main types: discoid lupus erythematosus (DLE), tumid lupus, and lupus panniculitis. Tumid lupus typically presents with juicy papules and plaques that heal without scarring, whereas lupus panniculitis involves the subcutaneous tissue, leading to painful subcutaneous nodules that heal with depression and atrophy.

Fumaderm® (dimethyl fumarate and ethyl hydrogen fumarate) has previously been used to treat CLE. See for example Klein, A., et al. (201 1), J Eur Acad Dermatol Venereol doi:

10.111 1/j.1468-3083.2011.04303.x. The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating CLE can be determined using animal models and in clinical trials. Suitable animal models for CLE are disclosed for example in Furukawa, F. (1997), Lupus 6(2): 193-202. Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia, also known as chronic lymphoid leukemia (CLL), is the most common type of adulthood leukemias. Leukemias are cancers of the white blood cells (leukocytes). CLL affects B cell lymphocytes. B cells originate in the bone marrow, develop in the lymph nodes, and normally fight infection by producing antibodies. In CLL, B cells grow out of control and accumulate in the bone marrow and blood, where they crowd out healthy blood cells. CLL is a stage of small lymphocytic lymphoma, a type of B-cell lymphoma, which presents primarily in the lymph nodes. Both are considered the same underlying disease, just with different appearances. CLL is a disease of adults, but, in rare cases, it can occur in teenagers and occasionally in children (inherited). Most (>75%) people newly diagnosed with CLL are over the age of 50, and the majority are men.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating chronic lymphocytic leukemia can be determined using animal models and in clinical trials.

Diabetic Retinopathy

Diabetic retinopathy (also sometimes referred to as diabetic retinitis) is retinopathy (damage to the retina) caused by complications of diabetes, which can eventually lead to blindness. A less serious type is called background retinopathy; a type that often progresses to blindness is called progressive retinopathy. Diabetic retinopathy is the most common diabetic eye disease and a leading cause of blindness in American adults. It is caused by changes in the blood vessels of the retina. In some people with diabetic retinopathy, blood vessels may swell and leak fluid. In other people, abnormal new blood vessels grow on the surface of the retina. Diabetic retinopathy usually affects both eyes.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating diabetic retinopathy can be determined using animal models and in clinical trials. Suitable animal models for diabetic retinopathy are disclosed for example in Robinson R., et al. (2012), Disease Models & Mechanisms 5(4):444-56.

Globoid Cell Leukodystrophy

Globoid cell leukodystrophy (also sometimes referred to as Krabbe Disease,

galactosylceramide lipidosis, globoid cell leukodystrophy, or Krabbe's leukodystrophy) is an inherited metabolic lysosomal storage disease that affects the muscles, vision, and mental abilities. It is life-threatening. It begins in infancy with irritability, fretfulness, and rigidity, followed by tonic seizures, convulsions, quadriplegia, blindness, deafness, dysphagia, and progressive mental deterioration. Pathologically, there is rapidly progressive cerebral demyelination and large globoid bodies in the white substance. In people with globoid cell leukodystrophy, the gene mutation affects an enzyme called galactocerebrosidase. Lack of this enzyme causes the buildup of a substance that damages cells that make myelin. This results in damage to the central nervous system. A person gets the disorder when he or she inherits a gene with the mutation from both parents. The disorder can appear soon after birth (early-onset globoid cell leukodystrophy) or in older children or adults (late-onset globoid cell

leukodystrophy). The disorder is rare; about 40 cases of globoid cell leukodystrophy are diagnosed in the United States each year.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating globoid cell leukodystrophy can be determined using animal models and in clinical trials. Suitable animal models for globoid cell leukodystrophy are disclosed for example in Gentner, B., et al. (2010) Science Translational Medicine 2(58); and Pellegatta, S., et al. (2006) Neurobiology of disease 21(2), 314-23.

Graft Versus Host Disease

Graft versus host disease (also sometimes referred to as graft versus host reaction) is a common complication following an allogeneic tissue transplant. It is commonly associated with stem cell or bone marrow transplant but the term also applies to other forms of tissue graft. Immune cells (white blood cells) in the tissue (the graft) recognize the recipient (the host) as "foreign". The transplanted immune cells then attack the host's body cells. Graft versus host disease can also occur after a blood transfusion if the blood products used have not been irradiated.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating graft versus host disease can be determined using animal models and in clinical trials. Suitable animal models for graft versus host disease are disclosed for example in Noth, R., et al. (2013) American Journal of Physiology - Gastrointestinal and Liver Physiology, 304:7 (G646-G654); and Ka, S.M. (2006) Nephrology Dialysis Transplantation, 21 :2 (288-298).

Hepatitis C Viral Infection

Hepatitis C (also sometimes referred to as HCV) is a viral disease of the liver caused by the hepatitis C virus, the most common form of post-transfusion hepatitis; it also follows parenteral drug abuse. It can also spread through sex with an infected person and from mother to baby during childbirth. Hepatitis C viral infection is a common acute sporadic hepatitis, with approximately 50% of acutely infected persons developing chronic hepatitis. Chronic infection is generally mild and asymptomatic, but cirrhosis or hepatocellular cancer may occur.

Most people who are infected with hepatitis C don't have any symptoms for years. A blood test can reveal the presence of the disease. Usually, hepatitis C does not get better by itself. The infection can last a lifetime and may lead to scarring of the liver or liver cancer. Medicines sometimes help, but side effects can be a problem. Serious cases sometimes require a liver transplant.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating hepatitis C viral infection can be determined using animal models and in clinical trials. Suitable animal models for hepatitis C are disclosed for example in Meuleman, P., et al. (201 1) Antimicrobial Agents and Chemotherapy 55(11), 5159-67; Moriishi, K., et al. (2007) Advanced Drug Delivery Reviews 59(12), 1213-21 ; and Pan, Q., et al. (2012) Hepatology 55(6), 1673-83.

Herpes Simplex Viral Infection

Herpes simplex is a group of acute infections caused by human herpesviruses 1 and 2, characterized by small fluid- filled vesicles on the skin or a mucous membrane with a raised erythematous base; it may be a primary infection or recurrent because of reactivation of a latent infection. Type 1 herpesvirus infections usually involve nongenital regions of the body, whereas type 2 infections are primarily on the genitals and surrounding areas, although there is overlap between the two types. Precipitating factors include fever, exposure to cold temperature or ultraviolet rays, sunburn, cutaneous or mucosal abrasions, emotional stress, and nerve injury. Oral herpes causes cold sores around the mouth or face. Genital herpes is a sexually transmitted disease (STD). It affects the genitals, buttocks or anal area. Other herpes infections can affect the eyes, skin, or other parts of the body. The virus can be dangerous in newborn babies or in people with weak immune systems.

Dimethyl fumarate has previously been administered to animals infected with the herpes simplex virus and improved the animals' herpes stromal keratitis. See for example Heiligenhaus, A., et al. (2005), Clinical and Experimental Immunology 142(1): 180-187; and Heiligenhaus, A., et al. (2004), Graefe's Archive for Clinical and Experimental Ophthalmology 242(10): 870-877. The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating herpes simplex viral infection can be determined using animal models and in clinical trials. Suitable animal models for herpes simplex are disclosed for example in Huang, W. Y., et al. (2010) Journal of General Virology 91(3), 591-98; and Prichard, M. N., et al. (2011) Antimicrobial Agents and

Chemotherapy 55(10), 4728-34.

Human Immunodeficiency Viral Infection

Human immunodeficiency virus (also sometimes referred to as HIV) is a virus of the genus Lentivirus, separable into two serotypes (HIV-1 and HIV-2), that is the etiologic agent of the acquired immunodeficiency syndrome (AIDS), the most advanced stage of infection with HIV. HIV-1, which comprises at least three subgroups (M, N, and O), is of worldwide distribution, while HIV-2 is largely confined to West Africa; transmission and manifestations are similar. The virus kills or damages the body's immune system cells. Transmission is commonly through unprotected sex with an infected person, by sharing drug needles or through contact with the blood of an infected person. Women can pass an HIV infection to their babies during pregnancy or childbirth.

Dimethyl fumarate and monomethyl fumarate have previously been suggested as a neuroprotectant in HIV patients. See for example Cross, S. A., et al. (2011), Journal of

Immunology 187(10): 5015-5025. The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating human immunodeficiency viral infection, and/or for use as a neuroprotectant in HIV patients, can be determined using animal models and in clinical trials. Suitable animal models for HIV are disclosed for example in Evans, D.T., et al. (2013), Curr Opin HIV AIDS doi: 10.1097/COH.0b013e328361cee8.

Lichen Planus

Lichen planus (also sometimes referred to as lichen ruber planus) is a chronic

mucocutaneous disease that affects the skin, tongue, nails and oral mucosa. The disease presents itself in the form of papules, lesions, or rashes. The name lichen planus refers to the dry and undulating, "lichen-like" appearance of affected skin. It is sometimes associated with oxidative stress as well as certain medications and diseases, however the underlying pathology is currently unknown. Lichen planus is characterized by an eruption of violet umbilicated, flat-topped, scaly papules with white lines or puncta (Wickham's striae), which may either be discrete or coalesce to form plaques or other shapes. Lichen planus has many types, including vesicular,

hypertrophic, atrophic, follicular, erosive and ulcerative, actinic, and erythematous; most resolve spontaneously, leaving residual hyperpigmentation and atrophy. Lichen planus-like lesions may also be caused by drugs or chemical substances.

A typical lichen planus rash displays with what clinicians call the "6 P's": well-defined pruritic, planar, purple, polygonal papules and plaques. The commonly affected sites are near the wrist and the ankle. The rash tends to heal with prominent blue-black or brownish discoloration that persists for a long time. Besides the typical lesions, many morphological varieties of the rash may occur. The presence of cutaneous lesions is not constant and may wax and wane over time. Oral lesions tend to last far longer than cutaneous lichen planus lesions.

Fumaderm® (dimethyl fumarate and ethyl hydrogen fumarate) and dimethyl fumarate alone have previously been used to treat lichen planus. See for example Guenther, C. H., et al. (2003), Annals of Pharmacotherapy 37(2): 234-236; and Klein, A., et al. (2012), Journal of the European Academy of Dermatology and Venereology 26(1 1): 1400-1406. The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating lichen planus can be determined using animal models and in clinical trials. A potential animal model for lichen planus is disclosed in Shi, G., et al. (2010) PLoS ONE 5: 10 Article Number: el3216. Macular Degeneration

Macular degeneration is a medical condition which usually affects older adults and results in a loss of vision in the center of the visual field (the macula) because of damage to the retina. It occurs in "dry" and "wet" forms. It is a major cause of blindness and visual impairment in older adults (>50 years). Macular degeneration can make it difficult or impossible to read or recognize faces, although enough peripheral vision remains to allow other activities of daily life.

Starting from the inside of the eye and going towards the back, the three main layers at the back of the eye are the retina, which contains the nerves; the choroid, which contains the blood supply; and the sclera, which is the white of the eye. The macula is the central area of the retina, which provides the most detailed central vision.

In the dry (nonexudative) form, cellular debris called drusen accumulates between the retina and the choroid, and the retina can become detached. In the wet (exudative) form, which is more severe, blood vessels grow up from the choroid behind the retina, and the retina can also become detached. It can be treated with laser coagulation, and with medication that stops and sometimes reverses the growth of blood vessels. Although some macular dystrophies affecting younger individuals are sometimes referred to as macular degeneration, the term generally refers to age-related macular degeneration.

Age-related macular degeneration begins with characteristic yellow deposits (drusen) in the macula, between the retinal pigment epithelium and the underlying choroid. Most people with these early changes (referred to as age-related maculopathy) have good vision. People with drusen can go on to develop advanced age-related macular degeneration (AMD). The risk is higher when the drusen are large and numerous and associated with disturbance in the pigmented cell layer under the macula. Large and soft drusen are related to elevated cholesterol deposits and may respond to cholesterol-lowering agents.

Based on in vitro testing on human retinal pigment epithelium cells, dimethyl fumarate has been suggested for use in treating macular degeneration. See for example Nelson, K. C, et al. (1999), Investigative Ophthalmology and Visual Science 40(9): 1927-1935; and Winkler, B. S., et al. (1999), Molecular vision 5: 32. The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating macular degeneration can be determined using animal models and in clinical trials. Suitable animal models for macular degeneration are disclosed for example in Catchpole, I., et al. (2013) PLoS ONE 8:6 Article Number: e65518; and Cruz-Guilloty, F., et al. (2013) International Journal of Inflammation Article Number: 503725.

Monomelic Amyotrophy

Monomelic amyotrophy is characterized by progressive degeneration and loss of motor neurons, the nerve cells in the brain and spinal cord that are responsible for controlling voluntary muscles. It is characterized by weakness and wasting in a single limb, usually an arm and hand rather than a foot and leg. There is no pain associated with the disease. Monomelic amyotrophy occurs in males between the ages of 15 and 25. Onset and progression are slow. The disease is seen most frequently in Asia, particularly in Japan and India; it is much less common in North America. In most cases, the cause is unknown, although there have been a few published reports linking monomelic amyotrophy to traumatic or radiation injury. There are also familial forms of monomelic amyotrophy. Diagnosis is made by physical exam and medical history.

Electromyography, a special recording technique that detects electrical activity in muscles, shows a loss of the nerve supply, or denervation, in the affected limb; MRI and CT scans may show muscle atrophy.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating monomelic amyotrophy can be determined using animal models and in clinical trials.

Necrobiosis Lipoidosis

Necrobiosis lipoidosis, also sometimes called necrobiosis lipoidica, is a degenerative disease of dermal connective tissue characterized by development of erythematous papules or nodules in the pretibial area and sometimes elsewhere, extending to form shiny yellow to red plaques that are covered with telangiectatic vessels and have a scaly, atrophic, depressed center. More than half of affected patients have diabetes; the clinical appearance, genetic background for diabetes, and histopathologic findings are similar in both diabetic and nondiabetic patients.

Fumaderm® (dimethyl fumarate and ethyl hydrogen fumarate) has previously been used to treat necrobiosis lipoidosis. See for example Eberle, F. C, et al. (2010), Acta Derm Venereol 90(1): 104-106; Gambichler, T., et al. (2003), Dermatology 207(4): 422-424; Kreuter, A., et al. (2005), British Journal of Dermatology 153(4): 802-807; and Wang, W. P., et al. (2007), Chinese Journal of Evidence-Based Medicine 7(11): 830-835. The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating necrobiosis lipoidosis can be determined using animal models and in clinical trials.

Neurodegeneration with Brain Iron Accumulation

Neurodegeneration with brain iron accumulation is a rare, inherited, neurological movement disorder characterized by an abnormal accumulation of iron in the brain and progressive degeneration of the nervous system. Symptoms, which vary greatly among patients and usually develop during childhood, may include slow writhing, distorting muscle contractions of the limbs, face, or trunk, choreoathetosis (involuntary, purposeless jerky muscle movements), muscle rigidity (uncontrolled tightness of the muscles), spasticity (sudden, involuntary muscle spasms), ataxia (inability to coordinate movements), confusion, disorientation, seizures, stupor, and dementia. Other less common symptoms may include painful muscle spasms, dysphasia (difficulty speaking), mental retardation, facial grimacing, dysarthria (poorly articulated speech), and visual impairment. Several genes have been found that cause the disease.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating

neurodegeneration with brain iron accumulation can be determined using animal models and in clinical trials.

Neuromyelitis Optica

Neuromyelitis optica (also referred to as Devic disease, optic neuroencephalomyelopathy, neuro-optic myelitis, and ophthalmoneuromyelitis) is an uncommon disease syndrome of the central nervous system that affects the optic nerves and spinal cord. The disease is marked by diminution of vision and possibly blindness, flaccid paralysis of the extremities, and sensory and genitourinary disturbances. Individuals with the disease develop optic neuritis, which causes pain in the eye and vision loss, and transverse myelitis, which causes weakness, numbness, and sometimes paralysis of the arms and legs, along with sensory disturbances and loss of bladder and bowel control. Neuromyelitis optica leads to loss of myelin, a fatty substance that surrounds nerve fibers and helps nerve signals move from cell to cell. The syndrome can also damage nerve fibers and leave areas of broken-down tissue. In the disease process of neuromyelitis optica, immune system cells and antibodies attack and destroy myelin cells in the optic nerves and the spinal cord. The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating neuromyelitis optica can be determined using animal models and in clinical trials. Suitable animal models for neuromyelitis optica are disclosed for example in Saadoun, S., et al. (2012) Annals of

Neurology 71(3), 323-33; and Tradtrantip, L., et al. (2012) Annals of Neurology 71(3), 314-22. Neurosarcoidosis

Neurosarcoidosis is a manifestation of sarcoidosis in the nervous system. Sarcoidosis is a chronic inflammatory disorder that typically occurs in adults between 20 and 40 years of age and primarily affects the lungs, but can also impact almost every other organ and system in the body. Neurosarcoidosis is characterized by inflammation and abnormal cell deposits in any part of the nervous system; the brain, spinal cord, or peripheral nerves. It most commonly occurs in the cranial and facial nerves, the hypothalamus (a specific area of the brain), and the pituitary gland. It is estimated to develop in 5 to 15 percent of those individuals who have sarcoidosis. Weakness of the facial muscles on one side of the face (Bell's palsy) is a common symptom of

neurosarcoidosis. The optic and auditory nerves can also become involved, causing vision and hearing impairments. It can cause headache, seizures, memory loss, hallucinations, irritability, agitation, and changes in mood and behavior. Neurosarcoidosis can appear in an acute, explosive fashion or start as a slow chronic illness.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating

neurosarcoidosis can be determined using animal models and in clinical trials.

Optic Neuritis

Optic neuritis is inflammation or demyelination of the optic nerve, the nerve that transmits light and visual images from the retina to the brain. Because the nerve is located behind ("retro") the globe of the eye, the condition is also known as retrobulbar neuritis. Optic neuritis is generally experienced as an acute blurring, graying (change in color saturation), or loss of vision, most often in only one eye. It is rare that both eyes are affected at the same time. There may or may not be pain in the affected eye. The pain, when it occurs, can be of several types; dull and aching, pressure-like, or sharp and piercing.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating optic neuritis can be determined using animal models and in clinical trials. Suitable animal models for optic neuritis are disclosed for example in Chaudhary, P., et al. (2011) Journal of neuroimmunology 233(1-2), 90-96; and Zhang, J., et al. (2011) International Journal of Ophthalmology 1 1(1), 43- 45.

Pareneoplastic Syndromes

Paraneoplastic syndromes are rare disorders that are triggered by an altered immune system response to a neoplasm. They are defined as clinical syndromes involving nonmetastatic systemic effects that accompany malignant disease. In a broad sense, these syndromes are collections of symptoms that result from substances produced by the tumor, and they occur remotely from the tumor itself. The disease is a symptom-complex arising in a cancer-bearing patient that cannot be explained by local or distant spread of the tumor. The symptoms may be endocrine, neuromuscular or musculoskeletal, cardiovascular, cutaneous, hematologic, gastrointestinal, renal, or miscellaneous in nature.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating

pareneoplastic syndromes can be determined using animal models and in clinical trials. Suitable animal models for pareneoplastic syndromes are disclosed for example in Barton, B. E., et al. (2000) Proceedings of the Society for Experimental Biology and Medicine 223(2), 190-97; and Diament, M. J., et al. (2006) Cancer Investigation 24(2), 126-31.

Pelizaeus-Merzbacher Disease

Pelizaeus-Merzbacher disease (also referred to as Merzbacher-Pelizaeus disease, familial centrolobar sclerosis, and Pelizaeus-Merzbacher sclerosis) is a rare, progressive, degenerative central nervous system disorder in which coordination, motor abilities, and intellectual function deteriorate. The disease is one of a group of gene-linked disorders known as the

leukodystrophies, which affect growth of the myelin sheath, the fatty covering that wraps around and protects nerve fibers in the brain. The disease is caused by a mutation in the gene that controls the production of a myelin protein called proteolipid protein-1 (PLP1). Pelizaeus- Merzbacher disease is inherited as an X-linked recessive trait; the affected individuals are male and the mothers are carriers of the PLP1 mutation. Severity and onset of the disease ranges widely, depending on the type of PLP1 mutation. Pelizaeus-Merzbacher disease is one of a spectrum of diseases associated with PLP1, which also includes Spastic Paraplegia Type 2 (SPG2). The PLP1 -related disorders span a continuum of neurologic symptoms that range from severe central nervous system involvement to progressive weakness and stiffness of the legs (SPG2). The disease occurs in early life and runs a slowly progressive course into adolescence or adulthood. It is marked by nystagmus, ataxia, tremor, choreoathetoid movements, parkinsonian facies, dysarthria, and mental deterioration. Pathologically, there is diffuse demyelination in the white substance of the brain, which may involve the brain stem, cerebellum, and spinal cord.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating Pelizaeus-

Merzbacher disease can be determined using animal models and in clinical trials. Suitable animal models for Pelizaeus-Merzbacher disease are disclosed for example in Wood, P. L., et al. (2011) Lipids in Health and Disease 10; and Yu, L. H., et al. (2012) Molecular Genetics and

Metabolism 106(1), 108-14.

Primary Lateral Sclerosis

Primary lateral sclerosis (also sometimes referred to as Erb's syndrome) is a rare neuromuscular disease with slowly progressive weakness in voluntary muscle movement.

Primary lateral sclerosis belongs to a group of disorders known as motor neuron diseases.

Primary lateral sclerosis affects the upper motor neurons (also called corticospinal neurons) in the arms, legs, and face. It occurs when nerve cells in the motor regions of the cerebral cortex (the thin layer of cells covering the brain which is responsible for most higher level mental functions) gradually degenerate, causing movements to be slow and effortful. Symptoms include weakness, muscle stiffness and spasticity, clumsiness, slowing of movement, and problems with balance and speech. Primary lateral sclerosis is more common in men than in women, with a varied gradual onset that generally occurs between ages 40 and 60. Primary lateral sclerosis progresses gradually over a number of years, or even decades. Scientists do not believe Primary lateral sclerosis has a simple hereditary cause.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating primary lateral sclerosis can be determined using animal models and in clinical trials.

Progressive Supranuclear Palsy

Progressive supranuclear palsy (also referred to as Steele-Richardson-Olszewski syndrome) is a rare brain disorder, having onset during the sixth decade, that causes serious and progressive problems with control of gait and balance, along with complex eye movement and thinking problems. The disease is characterized by supranuclear ophthalmoplegia, especially paralysis of the downward gaze, pseudobulbar palsy, dysarthria, dystonic rigidity of the neck and trunk, and dementia. One of the classic signs of the disease is an inability to aim the eyes properly, which occurs because of lesions in the area of the brain that coordinates eye movements. Some individuals describe this effect as a blurring. Affected individuals often show alterations of mood and behavior, including depression and apathy as well as progressive mild dementia.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating progressive supranuclear palsy can be determined using animal models and in clinical trials. Suitable animal models for progressive supranuclear palsy are disclosed for example in Lewis, J., et al. (2000) Nature genetics 25(4), 402-05; and O'Leary, J. C, et al. (2010) Molecular Neurodegeneration

5(1).

Schilder 's Disease

Schilder's disease (also referred to as encephalitis periaxialis diffusa, Flatau- Schilder disease and Schilder's encephalitis) is a rare progressive demyelinating disorder which usually begins in childhood. The disease is a subacute or chronic form of leukoencephalopathy of children and adolescents. Clinical symptoms include blindness, deafness, bilateral spasticity, aphasia, seizures, personality changes, poor attention, tremors, balance instability, incontinence, muscle weakness, headache, vomiting, speech impairment, progressive mental deterioration and dementia. There is massive destruction of the white substance of the cerebral hemispheres, cavity formation, and glial scarring.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating Schilder's

Disease can be determined using animal models and in clinical trials.

Subacute Necrotizing Myelopathy

Subacute necrotizing myelopathy (also sometimes referred to as subacute necrotizing encephalopathy or Leigh Disease) is a rare inherited neurometabolic disorder that affects the central nervous system. Subacute necrotizing myelopathy occurs in two forms: the infantile form, which may be the same as pyruvate carboxylase deficiency, is characterized by degeneration of gray matter with necrosis and capillary proliferation in the brain stem;

hypotonia, seizures, and dementia; anorexia and vomiting; slow or arrested development; and ocular and respiratory disorders. The disease can be caused by mutations in mitochondrial DNA or by deficiencies of an enzyme called pyruvate dehydrogenase. Symptoms of the disease usually progress rapidly. The earliest signs may be poor sucking ability, the loss of head control and motor skills. These symptoms may be accompanied by loss of appetite, vomiting, irritability, continuous crying, and seizures. As the disorder progresses, symptoms may also include generalized weakness, lack of muscle tone, and episodes of lactic acidosis, which can lead to impairment of respiratory and kidney function. Death usually occurs before age 3. The adult form usually first manifests as bilateral optic atrophy with central scotoma and colorblindness; then there is a quiescent period of up to 30 years; and then late symptoms appear such as ataxia, spastic paresis, clonic jerks, grand mal seizures, psychic lability, and mild dementia.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating subacute necrotizing myelopathy can be determined using animal models and in clinical trials. Suitable animal models for subacute necrotizing myelopathy are disclosed for example in Quintana, A., et al. (2012), Journal of Clinical Investigation 122:7 (2359-2368).

Susac's Syndrome

Susac's syndrome is a rare disorder characterized by impaired brain function

(encephalopathy), blockage (occlusion) of the arteries that supply blood to the retina (branched retinal arterial occlusion), and hearing loss. Two main forms of Susac's syndrome have been identified. In one form, encephalopathy is the main finding, in the other form, branched retinal arterial occlusion and hearing loss occur without signs of brain disease. The specific symptoms and severity of Susac's syndrome vary from one person to another. The encephalopathic form of Susac's syndrome often improves spontaneously even without treatment (self-limited); the other form is frequently a chronic disorder. Although considered rare, Susac's syndrome is being recognized more often worldwide and its true frequency in the general population is unknown. Susac's syndrome is considered an autoimmune endotheliopathy.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating Susac's syndrome can be determined using animal models and in clinical trials.

Transverse Myelitis

Transverse myelitis is a neurological disorder caused by inflammation across both sides of one level, or segment, of the spinal cord. The term myelitis refers to inflammation of the spinal cord; transverse simply describes the position of the inflammation, that is, across the width of the spinal cord. Attacks of inflammation can damage or destroy myelin, the fatty insulating substance that covers nerve cell fibers. This damage causes nervous system scars that interrupt communications between the nerves in the spinal cord and the rest of the body. Symptoms of transverse myelitis include a loss of spinal cord function over several hours to several weeks.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating transverse myelitis can be determined using animal models and in clinical trials.

Tumors

The term "tumor" is a commonly used synonym for a neoplasm, a solid or fluid-filled lesion, that may or may not be cystic, and may or may not be formed by an abnormal growth of neoplastic cells. Tumors typically appear as enlarged lesions. The term "tumor" is not synonymous with cancer, because it is not necessarily malignant. A tumor can be any one of benign, pre-malignant, or malignant and can also represent as a lesion without cancerous potential.

A tumor is typically caused by an abnormal proliferation of tissues, which may or may not be caused by one or more genetic mutations. Not all tumors cause a tumorous overgrowth of tissue, however.

In certain embodiments, a tumor is a solid tumor.

In certain embodiments, a the solid tumor is one of mamma carcinoma, colon carcinoma, melanoma, primary liver cell carcinoma, adenocarcinoma, kaposi's sarcoma, prostate carcinoma, multiple myeloma (plasmocytoma), Burkitt lymphoma, and Castleman tumor.

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating tumors can be determined using animal models and in clinical trials.

Zellweger Syndrome

Zellweger syndrome (also referred to as cerebrohepatorenal syndrome) is an autosomal recessive disorder characterized by craniofacial abnormalities, hypotonia, hepatomegaly, polycystic kidneys, jaundice, and death in early infancy, and associated with absence of peroxisomes in the liver and kidneys. The disease is one of a group of four related diseases called peroxisome biogenesis disorders, which are part of a larger group of diseases known as the leukodystrophies. These are inherited conditions that damage the white matter of the brain and also affect how the body metabolizes particular substances in the blood and organ tissues. The diseases are caused by defects in any one of 13 genes, termed PEX genes, required for the normal formation and function of peroxisomes. The disorders are divided into two groups:

Zellweger spectrum disorders and Rhizomelic Chondrodysplasia Punctua spectrum disorders. The Zellweger spectrum is comprised of three disorders that have considerable overlap of features. These include Zellweger syndrome (the most severe form), neonatal

adreno leukodystrophy, and Infantile Refsum disease (the least severe form).

The efficacy of compounds of Formulae (I), (II), (III), and (IV) for treating Zellweger syndrome can be determined using animal models and in clinical trials. Suitable animal models for Zellweger syndrome are disclosed for example in Mast, F. D., et al. (2011) DMM Disease Models and Mechanisms 4(5), 659-72; and Muller, C. C, et al. (2011) DMM Disease Models and Mechanisms 4(1), 104-19.

Others

Other diseases and conditions for which compounds of Formulae (I), (II), (III), and (IV) can be useful in treating include rheumatica, granuloma annulare, lupus, autoimmune carditis, eczema, sarcoidosis, and autoimmune diseases including acute disseminated encephalomyelitis, Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, Bechet's disease, celiac disease, Chagas disease, chronic obstructive pulmonary disease, Crohn's disease, dermatomyositis, diabetes mellitus type I, endometriosis,

Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hidradenitis suppurativea, Kawasaki disease, IgA neuropathy, idiopathic thrombocytopenic purpura, interstitial cystitis, lupus erythematosus, mixed connective tissue disease, morphea, multiple sclerosis, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, schizophrenia, scleroderma, Sjogren's syndrome, stiff person syndrome, temporal arteritis, ulcerative colitis, vasculitis, vitiligo, acute disseminated encephalomyelitis, myasthenia gravis, and Wegener's granulomatosis.

Administration

Compounds of Formulae (I), (II), (III), and (IV) and pharmaceutical compositions thereof may be administered orally or by any other appropriate route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings {e.g., oral mucosa, rectal, and intestinal mucosa, etc.). Other suitable routes of administration include, for example, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical.

Administration may be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., that may be used to administer a compound and/or pharmaceutical composition.

The amount of a compound of Formulae (I), (II), (III), and (IV) that will be effective in the treatment of a disease in a patient will depend, in part, on the nature of the condition and can be determined by standard clinical techniques known in the art. In addition, in vitro or in vivo assays may be employed to help identify optimal dosage ranges. A therapeutically effective amount of a compound of Formulae (I), (II), (III), and (IV) to be administered may also depend on, among other factors, the subject being treated, the weight of the subject, the severity of the disease, the manner of administration, and the judgment of the prescribing physician.

For systemic administration, a therapeutically effective dose may be estimated initially from in vitro assays. For example, a dose may be formulated in animal models to achieve a beneficial circulating composition concentration range. Initial doses may also be estimated from in vivo data, e.g., animal models, using techniques that are known in the art. Such information may be used to more accurately determine useful doses in humans. One having ordinary skill in the art may optimize administration to humans based on animal data.

A dose may be administered in a single dosage form or in multiple dosage forms. When multiple dosage forms are used the amount of compound contained within each dosage form may be the same or different. The amount of a compound of Formulae (I), (II), (III), and (IV) contained in a dose may depend on the route of administration and whether the disease in a patient is effectively treated by acute, chronic, or a combination of acute and chronic

administration.

In certain embodiments an administered dose is less than a toxic dose. Toxicity of the compositions described herein may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD ₅₀ (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. In certain embodiments, a compound or metabolite thereof may exhibit a high therapeutic index. The data obtained from these cell culture assays and animal studies may be used in formulating a dosage range that is not toxic for use in humans. A dose of a compound of Formulae (I), (II), (III), and (IV) may be within a range of circulating concentrations in for example the blood, plasma, or central nervous system, that include the effective dose and that exhibits little or no toxicity. A dose may vary within this range depending upon the dosage form employed and the route of administration utilized. In certain embodiments, an escalating dose may be administered.

Combination Therapy

Methods provided by the present disclosure further comprise administering one or more pharmaceutically active compounds in addition to a compound of Formula (I), (II), (III), or (IV). Such compounds may be provided to treat the same disease or a different disease than the disease being treated with the compound of Formula (I), (II), (III), or (IV).

In certain embodiments, a compound of Formula (I), (II), (III), or (IV) may be used in combination with at least one other therapeutic agent. In certain embodiments, a compound of Formula (I), (II), (III), or (IV) may be administered to a patient together with another compound for treating diseases and conditions involving immunological, autoimmune, and/or inflammatory processes including: multiple sclerosis, psoriasis, irritable bowel disorder, ulcerative colitis, arthritis, chronic obstructive pulmonary disease, asthma, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and others. In certain embodiments, a compound of Formula (I), (II), (III), or (IV) may be administered to a patient together with another compound for treating multiple sclerosis, psoriasis, irritable bowel disorder, ulcerative colitis, arthritis, chronic obstructive pulmonary disease, asthma, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis.

A compound of Formula (I), (II), (III), or (IV) and the at least one other therapeutic agent may act additively or, in certain embodiments, synergistically. The at least one additional therapeutic agent may be included in the same dosage form as a compound of Formula (I), (II), (III), or (IV) or may be provided in a separate dosage form. Methods provided by the present disclosure can further include, in addition to administering a compound of Formula (I), (II), (III), or (IV), administering one or more therapeutic agents effective for treating the same or different disease than the disease being treated by a compound of Formula (I), (II), (III), or (IV). Methods provided by the present disclosure include administration of a compound of Formula (I), (II), (III), or (IV) and one or more other therapeutic agents provided that the combined administration does not inhibit the therapeutic efficacy of the compound of Formula (I), (II), (III), or (IV), or any pharmacologically active metabolite thereof, and/or does not typically produce significant and/or substantial adverse combination effects.

In certain embodiments, dosage forms comprising a compound of Formula (I), (II), (III), or (IV) may be administered concurrently with the administration of another therapeutic agent, which may be part of the same dosage form as, or in a different dosage form than, that comprising a compound of Formula (I), (II), (III), or (IV). A compound of Formula (I), (II), (III), or (IV) may be administered prior to, or subsequent to, administration of another therapeutic agent. In certain embodiments, the combination therapy may comprise alternating between administering a compound of Formula (I), (II), (III), or (IV) and administering another therapeutic agent, e.g., to minimize adverse drug effects associated with a particular drug. When a compound of Formula (I), (II), (III), or (IV) is administered concurrently with another therapeutic agent that potentially may produce an adverse drug effect including, for example, toxicity, the other therapeutic agent may be administered at a dose that falls below the threshold at which the adverse drug effect is elicited.

In certain embodiments, dosage forms comprising a compound of Formula (I), (II), (III), or (IV) may be administered with one or more substances to enhance, modulate and/or control release, bioavailability, therapeutic efficacy, therapeutic potency, stability, and the like of a compound of Formula (I), (II), (III), or (IV). For example, to enhance the therapeutic efficacy of a compound of Formula (I), (II), (III), or (IV), the compound of Formula (I), (II), (III), or (IV) may be co-administered with, or a dosage form comprising a compound of Formula (I), (II), (III), or (IV) may comprise, one or more active agents to increase the absorption or diffusion of a compound of Formula (I), (II), (III), or (IV) from the gastrointestinal tract to the systemic circulation, or to inhibit degradation of the compound of Formula (I), (II), (III), or (IV) in the blood of a patient. In certain embodiments, a compound of Formula (I), (II), (III), or (IV) may be co-administered with an active agent having pharmacological effects that enhance the therapeutic efficacy of a compound of Formula (I), (II), (III), or (IV). In certain embodiments, a compound of Formula (I), (II), (III), or (IV), or a pharmaceutical composition thereof may be administered to a patient for treating psoriasis in combination with a therapy or another therapeutic agent known or believed to be effective in treating psoriasis. Drugs useful for treating psoriasis include, for example, steroids such as flurandrenolide, fluocinonide, alclometasone, amcinonide, desonide, halcinonide, triamcinolone, clobetasol, clocortolone, mometasone, desoximetasone, and halobetasol; anti-rheumatics such as etanercept, infiximab, and adalimumab; immunosuppressive agents such as cyclosporine, alefacept, and efalizumab; psoralens such as methoxsalen; and other such as calcipotriene, methotrexate, hydrocortisone/pramoxine, acitretin, betamethasone/calcipotriene, tazaraotene, benzocaine/pyrilamine/zinc oxide, and ustekinumab.

In certain embodiments, a compound of Formula (I), (II), (III), or (IV), or a

pharmaceutical composition thereof may be administered to a patient for treating inflammatory arthritis such as rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis in combination with a therapy or another therapeutic agent known or believed to be effective in treating inflammatory arthritis such as rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis.

Drugs useful for treating rheumatoid arthritis include, for example, non-steroidal antiinflammatory agents such as ibuprofen, ketoprofen, salicylate, diclofenac, nabumetone, naproxen, meloxicam, sulindac, flurbiprofen, indomethacin, tolmetin, piroxicam, fenoprofen, oxaprozin, and etodolac; antiheumatics such as entanercept, adalimumab, infliximab, hydroxychloroquine, leflunomide, azathioprine, penicillamine, methotrexate, anakinra, auranofin, rituximab, aurothioglucose, tocilizumab, and golimumab; COX-2 inhibitors such as celecoxib and vadecoxib; corticosteroids such as triamcinolone; glucocorticoids such as methylprednisolone and prednisone; and others such as sulfasalazine.

Drugs useful for treating juvenile rheumatoid arthritis include, for example, adalimumab, abatacept, and infliximab.

Drugs useful for treating psoriatic arthritis include, for example, etanercept, adalimumab, triamcinolone, cortisone, infliximab, and golimumab. Drags useful for treating ankylosing spondylitis include, for example, adalimumab, celecoxib, diclofenac, etanercept, golimumab, indomethacin infliximab, naptoxen, olsalazine, salicylates, sulfindac, and triamcinolone.

In certain embodiments, a compound of Formula (I), (II), (III), or (IV), or a

pharmaceutical composition thereof, may be administered to a patient for treating psoriatic arthritis in combination with a therapy or another therapeutic agent known or believed to be effective in treating psioriatic arthritis. Drags useful for treating psioriatic arthritis include, for example, etanercept, adalimumab, triamcinolone, cortisone, infliximab, and golimumab.

In certain embodiments, a compound of Formula (I), (II), (III), or (IV), or a

pharmaceutical composition thereof, may be administered to a patient for treating autoimmune diseases such as lupus in combination with a therapy or another therapeutic agent known or believed to be effective in treating autoimmune diseases such as lupus. Drags useful for treating lupus include, for example, hydroxychloroquine, triamcinolone, salicylate, azathioprine, and abetimus.

In certain embodiments, a compound of Formula (I), (II), (III), or (IV), or a

pharmaceutical composition thereof may be administered to a patient for treating multiple sclerosis in combination with a therapy or another therapeutic agent known or believed to be effective in treating multiple sclerosis. Drags useful for treating multiple sclerosis include, for example, interferon β-la, interferon β-lb, glatiramer, modafinil, azathioprine, prednisolone, mycophenolate mofetil, mitoxantrone, and natalizumab. Other examples of drags useful for treating MS include, for example, corticosteroids such as methylprednisolone; IFN-β such as IFN-p ia and IFN-p ib; glatiramer acetate; monoclonal antibodies that bind to the very late antigen-4 (VLA-4) integrin such as natalizumab; immunomodulatory agents such as FTY 720 sphinogosie-1 phosphate modulator and COX-2 inhibitors such as BW755c, piroxicam, and phenidone; and neuroprotective treatments including inhibitors of glutamate excitotoxicity and iNOS, free-radical scavengers, and cationic channel blockers; memantine; AMPA antagonists such as topiramate; and glycine-site NMD A antagonists.

In certain embodiments, a compound of Formula (I), (II), (III), or (IV), or a

pharmaceutical composition thereof may be administered to a patient for treating inflammatory bowel disease in combination with a therapy or another therapeutic agent known or believed to be effective in treating inflammatory bowel disease. Drugs useful for treating inflammatory bowel disease include, for example, cromolyn and mercaptopurine; and more particularly for treating Crohn's disease include certolizumab, budesonide, azathioprine, sulfasalazine, metronidazole, adalimumab, mercaptopurine, infliximab, mesalamine, and natalizumab; and for treating ulcerative colitis include balsalazide, infliximab, azathioprine, mesalamine, and cyclosporine.

In certain embodiments, a compound of Formula (I), (II), (III), or a pharmaceutical composition thereof may be administered to a patient for treating irritable bowel syndrome in combination with a therapy or another therapeutic agent known or believed to be effective in treating irritable bowel syndrome. Drugs useful for treating irritable bowel syndrome include, for example, lactobacillus acidophilus, dicylmine, atropine, hyoscyamine, phenobarbital, scopolamine, venlafaxine, chloridazepoxide, clidinium, alosetron, psyllium, cholestyramine, rifaximin, and tegaserod.

In certain embodiments, compounds of Formula (I), (II), (III), or (IV) and pharmaceutical compositions thereof may be administered to a patient for treating asthma in combination with a therapy or another therapeutic agent known or believed to be effective in treating asthma, or in certain embodiments, a disease, disorder, or condition associated with asthma. Examples of drugs useful in treating asthma include, for example, albuterol, aminophylline, beclomethasone, bitolterol, budesonide, cromolyn, ephedrine, epinephrine, flunisolide, fluticasone, formoterol, hydrocortisone, isoproterenol, levalbuterol, methylprednisolone, prednisolone, prednisone, pirbuterol, metaproterenol, racepinephrine, omalizumab, oxytriphylline, mometusone, montelukast, nedocromil, oxtriphylline, pirbuterol, salmeterol, terbutaline, theophylline, triamcinolone, zafirlukast, and zileuton.

In certain embodiments, compounds of Formula (I), (II), (III), or (IV) and pharmaceutical compositions thereof may be administered to a patient for treating chronic obstructive pulmonary disease in combination with a therapy or another therapeutic agent known or believed to be effective in treating chronic obstructive pulmonary disease, or in certain embodiments, a disease, disorder, or condition associated with chronic obstructive pulmonary disease. Examples of drugs useful for treating chronic obstructive pulmonary disease include, for example, albuterol, arformoterol, azithromycin, bitolterol, epinephrine, fluticasone, formoterol, ipratropium, isoproterenol, levabuterol, metaproterenol, pirbuterol, racepinephrine, salmeterol, and tiotropium. Useful drugs for treating chronic obstructive pulmonary disease further include, for example, bronchodialators such as β2 agonists such as salbutamol, bambuterol, clenbuterol, fenoterol, and formoterol; M3 antimuscarinics such as ipratropium; leukotriene antagonists such as montelukast, pranlukast, and zafirlukast; cromones such as cromoglicate and nedocromil; xanthines such as theophylline; corticosteroids such as beclomethasone, mometasone, and fluticasone; and TNF antagonists such as infliximab, adalimumab, and etanercept. Other treatments for chronic obstructive pulmonary disease include oxygen therapy, and pulmonary rehabilitation.

In certain embodiments, compounds of Formula (I), (II), (III), or (IV) and pharmaceutical compositions thereof may be administered to a patient for treating angiogenesis in combination with a therapy or another therapeutic agent known or believed to be effective in treating angiogenesis. Useful drugs for treating angiogenesis include, for example, angiostatin, endostatin, vitaxin, bevacizumab, thalidomide, batimastat, marimastat, carboxyamidotraizole, TNP-470, CMlOl, IFN-a, IL-12, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR, angiostatic steroids, cartilage-derived angiogenesis inhibitory factor, matrix metalloproteinase inhibitors, 2-methoxyestradiol, tecogalan, thrombospondin, prolactin, αγβ ₃ inhibitors, and lino mi de.

In certain embodiments, compounds of Formula (I), (II), (III), or (IV) and pharmaceutical compositions thereof may be administered to a patient for treating transplant rejection in combination with a therapy or another therapeutic agent known or believed to be effective in treating transplant rejection. Useful drugs for treating transplant rejection include, for example, calcineurin inhibitors such as cyclosporine and tacrolimus, mTOR inhibitors such as sirolimus and everolimus, anti-proliferatives such as azathioprine and mycophenolic acid; monoclonal anti-IL2Ra receptor antibodies including basiliximab and daclizumab; and polyclonal anti-T-cell antibodies including anti-thymocyte globulin and anti-lymphocyte globulin.

In certain embodiments, compounds of Formula (I), (II), (III), or (IV) and pharmaceutical compositions thereof may be administered to a patient for treating transplantation rejection in combination with a therapy or another therapeutic agent known or believed to be effective in treating transplantation rejection. Examples of drugs useful in transplantation rejection include, for example, corticosteroids such as dexamethasone, prednisolone, and prednisone; globulins such as antilymphocyte globulin and antithymocyte globulin; macrolide immunosuppressants such as sirolimus, tacrolimus, and everolimus; mitotic inhibitors such as azathiprine,

cylophosphamide, and methotrexate; monoclonal antibodies such as basiliximab, daclizumab, infliximab, muromonoab; fungal metabolites such as cyclosporine; and others such as glatiramer and mycophenolate.

In certain embodiments, compounds of Formula (I), (II), (III), or (IV) and pharmaceutical compositions thereof may be administered to a patient for treating cardiac insufficiency in combination with a therapy or another therapeutic agent known or believed to be effective in treating cardiac insufficiency. Useful drugs for treating cardiac insufficiency include, for example, antitensin-modulating agents, diuretics such as furosemide, bumetanie,

hydrochlorothiazide, chlorthalidone, chlorthiazide, spironolactone, eplerenone: beta blockers such as bisoprolol, carvedilol, and metroprolol; positive inotropes such as digoxin, milrinone, and dobutamine; alternative vasodilators such as isosorbide dinitrate/hydralazine; aldosterone receptor antagonists; recombinant neuroendocrine hormones such as nesiritide; and vasopressin receptor antagonists such as tolvaptan and conivaptan.

In certain embodiments, compounds of Formula (I), (II), (III), or (IV) and pharmaceutical compositions thereof may be administered to a patient for treating a mitochondrial disease such as a neurodegenerative disease in combination with a therapy or another therapeutic agent known or believed to be effective in treating a mitochondrial disease such as a neurodegenerative disease. In certain embodiments, a neurodegenerative disease is chosen from Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.

Therapeutic agents useful for treating Parkinson's disease include, for example, dopamine precursors such levodopa, dopamine agonists such as bromocriptine, pergolide, pramipexole, and ropinirole, MAO-B inhibitors such as selegiline, anticholinergic drugs such as benztropine, trihexyphenidyl, tricyclic antidepressants such as amitriptyline, amoxapine, clomipramine, desipramine, doxepin, imipramine, maprotiline, nortriptyline, protriptyline, amantadine, and trimipramine, some antihistamines such as diphenhydramine; antiviral drugs such as amantadine; and beta blockers such as propranolol. Useful drags for treating Alzheimer's disease include, for example, raloxifene, vitamin E, donepezil, tacrine, rivastigmine, galantamine, and memantine.

Useful drags for treating symptoms of Huntington's disease include, for example, antipsychotics such as haloperidol, chlorpromazine and olanzapine to control hallucinations, delusions and violent outbursts; antidepressants such as fluoxetine, sertraline, and nortriptyline to control depression and obsessive-compulsive behavior; tranquilizers such as benzodiazepines, paroxetine, venflaxin and beta-blockers to control anxiety and chorea; mood stabilizers such as lithium, valproate, and carbamzepine to control mania and bipolar disorder; and botulinum toxin to control dystonia and jaw clenching. Useful drags for treating symptoms of Huntington's disease further include selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, paroxetine, sertraline, escitalopram, citalopram, fluvosamine; norepinephrine; serotonin reuptake inhibitors ( SRI) such as venlafaxine and duloxetine; benzodiazepines such as clonazepam, alprazolam, diazepam, and lorazepam; tricyclic antidepressants such as amitriptyline, nortriptyline, and imipramine; atypical antidepressants such as busipirone, bupriopion, and mirtazepine for treating the symptoms of anxiety and depression; atomoxetine,

dextroamphetamine, and modafinil for treating apathy symptoms; amantadine, memantine, and tetrabenazine for treating chorea symptoms; citalopram, atomoxetine, memantine, rivastigmine, and donepezil for treating cognitive symptoms; lorazepam and trazedone for treating insomnia; valproate, carbamazepine and lamotrigine for treating symptoms of irritability; SSRI

antidepressants such as fluoxetine, paroxetine, sertaline, and fluvoxamine; NSRI antidpressants such as venlafaxine; others such as mirtazepine, clomipramine, lomotrigine, gabapentin, valproate, carbamazepine, olanzapine, rispiridone, and quetiapine for treating symptoms of obsessive-compulsive disorder; haloperidol, quetiapine, clozapine, risperidone, olanzapine, ziprasidone, and aripiprazole for treating psychosis; and pramipexole, levodopa and amantadine for treating rigidity.

Useful drags for treating ALS include, for example, riluzole. Other drags of potential use in treating ALS include, for example, memantine, tamoxifen, thalidomide, ceftriaxone, sodium phenyl butyrate, celecoxib, glatiramer acetate, busipirone, creatine, minocycline, coenzyme Q10, oxandrolone, IGF- 1 , topiramate, xaliproden, and indinavir. Drags such as baclofen and diazepam can be useful in treating spasticity associated with ALS. In certain embodiments, a compound of Formula (I), (II), (III), or (IV), or a pharmaceutical composition thereof may be administered to a patient in combination with a therapy or another therapeutic agent known or believed to be effective in inhibiting TNF function.

Examples of drugs known to inhibit TNF function include, for example, infliximab, adalimumab, etanercept, certolizumab, goliimumab, pentoxifylline, quanylhydrozone, thalidomide, flavonoids such as narigenin, resveratol and quecetin, alkaloids such as lycorine, terpenes such as acanthoic acid, fatty acids such as 13-HOA, and retinoids such as retinoic acid.

Examples The following examples describe in detail the synthesis of mo no methyl and monoethyl fumarates of Formula (I), (II), (III), or (IV), properties of compounds of Formula (I), (II), (III), or (IV), and uses of compounds of Formula (I), (II), (III), or (IV). It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the disclosure.

General Experimental Protocols

All reagents and solvents that were purchased from commercial suppliers were used without further purification or manipulation procedures.

Proton NMR (400 MHz) and carbon NMR spectra (125 MHz) were recorded on a Varian AS 400 NMR spectrometer equipped with an autosampler and data processing software. CDC1 ₃ (99.8% D), D ₂ 0 (99.9% D), or MeOH-d ₄ (99.8+% D), and acetonitrile-d ₃ were used as solvents unless otherwise noted. The CHC1 ₃ , DMSO-ds, or MeOH-d ₃ solvent signals were used for calibration of the individual spectra. Analytical thin layer chromatography (TLC) was performed using a Whatman, Schleicher & Schuell T LC and MK6F silica gel plates (2.5x7.5 cm, 250 μηι layer thickness). Melting points were recorded in glass capillaries using a Stanford Research Systems (SRS) Optimelt Automated Melting Point System, S/N 78047. Analytical LC/MS was performed on a Waters 2790 separation module equipped with a Waters Micromass QZ mass spectrometer, a Waters 996 photodiode detector, and a Merck Chromolith UM2072-027 or Phenomenex Luna C-18 analytical column. Mass-guided preparative HPLC purification of final compounds was performed using an instrument equipped with a Waters 600 controller, ZMD Micromass spectrometer, a Waters 2996 photodiode array detector, and a Waters 2700 Sample Manager. Acetonitrile/water gradients containing 0.05% formic acid were used as eluents in both analytical and preparative HPLC experiments. Compound isolation from aqueous solvent mixtures, e.g., acetonitrile/water/0.05% formic acid, was accomplished by primary lyophilization (freeze drying) of the frozen solutions under reduced pressure at room temperature using manifold freeze dryers such as a Heto Drywinner DW 6-85-1, a Heto FD4, or a VIRTIS

Freezemobile 25 ES equipped with high vacuum pumps. When the isolated compound had ionizable functional groups such as an amino group or a carboxylic acid, lyophilization was performed in the presence of a slight excess of one molar (1 M) hydrochloric acid to yield the purified compounds as the corresponding hydrochloride salts (HCl-salts) or the corresponding protonated free carboxylic acids. When the isolated compound had ionizable functional groups such as a carboxylic acid, lyophilization was performed in the presence of equimolar amounts of sodium hydrogen carbonate (NaHC0 ₃ ) to yield the purified compounds as the corresponding sodium salts (Na-salts). Optionally, the isolated materials were further purified by flush silica gel column chromatography, optionally employing Biotage pre-packed silica gel cartridges. Suitable organic solvents such as ethyl acetate (EtOAc), hexane (Hxn), n-heptane (Hptn), or mixtures and/or gradients thereof were used as eluents to yield the target compounds as colorless, viscous oils or solids after evaporation of the solvents. Chemical names were generated with the

Chemistry 4-D Draw Pro Version 7.01c (Draw Chemical Structures Intelligently© 1993-2002) from Chemlnnovation Software, Inc., San Diego, USA).

Non-commercially available appropriately functionalized or substituted, hydroxyl compounds and N-alkyl-N-alkyloxycarbonylaminomethyl chloride derivatives were synthesized from commercially available starting materials, and by adapting methods well known in the art. General Synthetic Procedures:

General Procedure A: Activation of Carboxylic Acid Derivatives with Dehydration Agents for Alcoholysis or aminolysis.

(2E)-3-(Methoxycarbonyl)prop-2-enoic acid (methyl hydrogen fumarate, MHF), 2-[(2E)- 3-(methoxycarbonyl)prop-2-enoyloxy]acetic acid (23) or 2-[(2E)-3-(methoxycarbonyl)prop-2- enoyloxy]propanoic acid (24), (1.0 equivalents) are reacted at temperature from ca. 0° C. (ice bath) to room temperature with 1.0-1.5 equivalents of a carbodiimide dehydration agent such as l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (ED AC, EDC), N,N-diisopropylcarbodiimide (DIC), N,N-dicyclohexylcarbodiimide (DCC) in an inert solvent such as dichloromethane (DCM), N,N-dimethylformamide (DMF), N-methylpyrrolidone ( MP), or N,N- dimethylacetamide (DMA, DMAc) (ca. 3 mL/mmol). 1.0-1.5 Equivalents of an appropriately functionalized hydroxyl compound dissolved in the same solvent and, optionally, in the presence of a catalytic or stoichiometric amount of 4-(N,N-dimethylaminopyridine (DMAP) is added at a temperature from ca. 0° C. to room temperature. When the amine is a salt form, an equimolar amount of an organic tertiary base, such as triethylamine (TEA), or diisopropylethylamine (DIEA) may be added to free the amine base prior to the coupling step. The reaction mixture is stirred for 4 to 12 hours at room temperature. Optionally the organic solvents are removed under reduced pressure using a rotary evaporator and the residue diluted with an appropriate extraction solvent such as diethyl ether (Et ₂ 0), methyl tert-butyl ether (MTBE), ethyl acetate (EtOAc), or others. After phase separation, the aqueous phase is extracted several times with the same solvent. The combined organic extracts are washed with water, brine, and dried over anhydrous magnesium sulfate (MgSO/ _t ). After filtration, the organic solvents are removed under reduced pressure using a rotary evaporator. If required, the crude reaction products are further purified by well-known purification techniques such as silica gel flash column chromatography (i.e., Biotage), mass-guided reversed-phase preparative HPLC/lyophilization, precipitation, or crystallization.

General Procedure B: Hydrochloride salt formation

Alkyl-amino methyl (2E)but-2-ene-l,4-dioate derivative was dissolved in methyl tert- butyl ether. The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to precipitate/crystallize out as off- white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert- vXy\ ether. The filter-cake was dried under vacuum oven at 30°C to afford the title compound as off-white solid. General Procedure C: Nucleophilic Substitution of iV-alkyl-iV- alkyloxycarbonylaminomethyl chloride derivatives with Monomethyl Fumarate

(2E)-3-(Methoxycarbonyl)prop-2-enoic acid (methyl hydrogen fumarate, MHF), (2E)-3- (/er/-butoxycarbonyl)prop-2-enoic acid (/er/-butyl hydrogen fumarate), or fumaric acid (FA) (1.0 equivalents) is dissolved in 5-10 mL/3.0 mmol of an inert solvent such as N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA, DMAc), acetonitrile (MeCN), dimethylsulfoxide (DMSO), tetrahydrofuran (THF), toluene, or mixtures thereof. To the solution, 0.8 to 1.2 equivalents of an appropriate inorganic base such as cesium hydrogen carbonate (CsHC0 ₃ ), cesium carbonate (CS ₂ CO ₃ ), or potassium carbonate (K ₂ CO ₃ ) is added. Alternatively, 0.8 b is 1.2 equivalents of a silver salt such silver(I) oxide (Ag ₂ 0) or silver(I) carbonate (Ag ₂ C0 ₃ ); an organic secondary or tertiary base such as dicyclohexylamine (DCHA), triethylamine (TEA), diisopropylethylamine (DIEA), tetrabutylammonium hydroxide (TBAOH), amidine; or a guanidine-based base such as l,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU), or 1,1 ,3,3-tetramethylguanidine (TMG), can be employed. The corresponding alkali, silver, di-, tri- and tetraalkylammonium, amidine, or guanide salt of monoalkyl fumarate can also be preformed. The solution is stirred for 10-60 min at room temperature followed by addition of 0.8- 1.2 equivalents of an appropriately

functionalized N-alkyl-N-alkyloxycarbonylaminomethyl chloride derivative. The reaction mixture is stirred overnight at a temperature between 40 to 100° C. After cooling to room temperature, insolubles can optionally be filtered off and the reaction mixture diluted with one molar (1.0 M) hydrochloric acid (HQ) and an appropriate organic solvent such as methyl tert- butyl ether (MTBE), diethyl ether (Et ₂ 0), ethylacetate (EtOAc), or mixtures thereof. After phase separation, the aqueous phase is extracted several times with the same solvent. The combined organic extracts are washed with water, brine, and dried over anhydrous magnesium sulfate (MgSO/t). After filtration, the organic solvents are removed under reduced pressure using a rotary evaporator. If required, the crude reaction products are further purified by well known purification techniques such as silica gel flash column chromatography (i.e., Biotage), mass- guided reversed-phase preparative HPLC/lyophilization, precipitation, or crystallization. Example 1

2-(dimethylamino)ethyl methyl (2E)but-2-ene-l,4-dioate (3)

Following general Procedure A, methyl hydrogen fumarate (MHF) (13g, 0.1 mol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (20 g, 0.1 mol) in 100 mL of dichloromethane (DCM) at ca. 0°C. 2-(dimethylamino) ethanol (8.9 g, 0.1 mol) and 4-N,N-dimethylaminopyridine (DMAP) (1 g, 0.008 mol) were added to the activated carboxylic acid. After synthesis, 12.5 g (62.2 % yield) of the title compound was isolated as a viscous-oil.

Example 2

2-(dimethylamino)ethyl methyl (2E)but-2-ene-l,4-dioate HC1 (4)

Following general Procedure B, 2-(dimethylamino)ethyl methyl (2E)but-2-ene-l ,4-dioate (12.0 g, 0.059 mol) was dissolved in methyl tert-buty\ ether (100 mL). The resulting clear reaction mixture was cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product started to precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (100 mL). The filter-cake was dried under vacuum oven at 30°C to yield 9.0 g (63.6% yield) of the title compound as an off-white solid.

1H NMR (CDC1 ₃ , 400 MHz): δ 12.98 (broad s,-N ⁺ Me ₂ H, IH), 6.98 (d, J= 16 Hz, IH), 6.88 (d, J= 16 Hz, IH), 4.74 (t, J= 4.8 Hz, 2H), 3.82 (s, 3H), 3.44 (m, 2H), 2.93 (s, 3H), 2.91 (s, 3H). MS (ESI): m/z 203.10 (M+H) ⁺ . Melting point: 121.1°C. Example 3

3-(dimethylamino)propyl methyl (2E)but-2-ene-l,4-dioate (5)

Following general Procedure A, methyl hydrogen fumarate (MHF) (13g, 0.1 mol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (20 g, 0.1 mol) in 100 mL of dichloromethane (DCM) at ca. 0°C. 3-(dimethylamino) propanol (10.3 g, 0.1 mol) and 4-N,N-dimethylaminopyridine (DMAP) (1 g, 0.008 mol) were added to the activated carboxylic acid. After synthesis, 10.7 g (49.7 % yield) of the title compound was isolated as a viscous-oil.

Example 4

3-(dimethylamino)propyl methyl (2E)but-2-ene-l ,4-dioate HC1 (6)

Following general Procedure B, 3-(dimethylamino)propyl methyl (2E)but-2-ene-l,4- dioate (10.0 g, 0.047 mol) was dissolved in methyl tert-buty\ ether (100 mL). The resulting clear reaction mixture was cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product started to precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (100 mL). The filter-cake was dried under vacuum oven at 30°C to yield 5.0 g (43.1% yield) of the title compound as an off-white solid. 1H NMR (CDC1 ₃ , 400 MHz): δ 12.45 (broad s,-N ⁺ Me ₂ H, IH), 6.88 (d, J= 15.6 Hz, IH), 6.83 (d, J= 15.6 Hz, IH), 4.35 (t, J= 5.6 Hz, 2H), 3.82 (s, 3H), 3.18 (m, 2H), 2.89 (s, 3H), 2.87 (s, 3H), 2.34 (m, 2H). MS (ESI): m z 217.12 (M+H) ⁺ . Melting point: 139.2°C. Example 5

4-(dimethylamino)butyl methyl (2E)but-2-ene- 1 ,4-dioate (7)

Following general Procedure A, methyl hydrogen fumarate (MHF) (13g, 0.1 mol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (20 g, 0.1 mol) in 100 mL of dichloromethane (DCM) at ca. 0°C. 4-(dimethylamino) butanol (11.7 g, 0.1 mol) and 4-N,N-dimethylaminopyridine (DMAP) (1 g, 0.008 mol) were added to the activated carboxylic acid. After synthesis, 10.4 g (45.4 % yield) of the title compound was isolated as a viscous-oil.

Example 6

4-(dimethylamino)butyl methyl (2E)but-2-ene-l,4-dioate HC1 (8)

Following general Procedure B, 4-(dimethylamino)butyl methyl (2E)but-2-ene-l,4-dioate (10.0 g, 0.043 mol) was dissolved in methyl tert-buty\ ether (100 mL). The resulting clear reaction mixture was cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product started to precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (100 mL). The filter-cake was dried under vacuum oven at 30°C to yield 10.0 g (86.2% yield) of the title compound as an off-white solid.

1H NMR (CDC1 ₃ , 400 MHz): δ 12.29 (broad s,-N ⁺ Me ₂ H, 1H), 6.80-6.88 (m, 2H), 4.25 (t, J= 6.4 Hz, 2H), 3.82 (s, 3H), 3.08 (m, 2H), 2.86 (s, 3H), 2.84 (s, 3H), 1.98 (m, 2H), 1.82 (m, 2H). MS (ESI): m/z 231.13 (M+H) ⁺ . Melting point: 81.4°C. Example 7

5-(dimethylamino)pentyl methyl (2E)but-2-ene-l,4-dioate (9)

Following general Procedure A, methyl hydrogen fumarate (MHF) (1.3g, 10 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (2.0 g, 10 mmol) in 10 mL of dichloromethane (DCM) at ca. 0°C (ice bath). 5-(dimethylamino) pentanol (11.7 g, 0.1 mol) and 4-N,N-dimethylaminopyridine (DMAP) (50 mg) were added to the activated carboxylic acid. After synthesis, 1.0 g (41.6% yield) of the title compound was isolated as a viscous-oil.

Example 8

5-(dimethylamino)pentyl methyl (2E)but-2-ene-l ,4-dioate HC1

Following general Procedure B, 5-(dimethylamino)pentyl methyl (2E)but-2-ene-l,4- dioate (243 mg, 1 mmol) was dissolved in methyl tert-buty\ ether (10 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to

precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (2 mL). The filter-cake was dried under vacuum oven at 30°C to yield 100 mg (35.8% yield) of the title compound as an off-white solid. 1H NMR (CDC1 ₃ , 400 MHz): δ 12.50 (broad s,-N+Me ₂ H, 1H), 6.80-6.88 (m , 2H), 4.22 (t, J= 6.4 Hz, 2H), 3.82 (s, 3H), 3.0 (m, 2H), 2.82 (s, 3H), 2.81 (s, 3H), 1.93 (m, 2H), 1.76 (m, 2H), 1.48 (m, 2H). MS (ESI): m/z 245.15 (M ⁺ H) ⁺ . Melting point: 1 19.3°C. Example 9

6-(dimethylamino)hexyl methyl (2E)but-2-ene-l,4-dioate (11)

Following general Procedure A, methyl hydrogen fumarate (MHF) (13g, 0.1 mol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (20 g, 0.1 mol) in 100 mL of dichloromethane (DCM) at ca. 0°C. 6-(dimethylamino) hexanol (14.5 g, 0.1 mol) and 4-N,N-dimethylaminopyridine (DMAP) (1 g, 0.008 mol) were added to the activated carboxylic acid. After synthesis, 13.2 g (51.3 % yield) of the title compound was isolated as a viscous-oil.

Example 10

6-(dimethylamino)hexyl methyl (2E)but-2-ene-l,4-dioate HC1 (12)

Following general Procedure B, 6-(dimethylamino)hexyl methyl (2E)but-2-ene-l,4- dioate (13.0 g, 0.05 mol) was dissolved in methyl tert-buty\ ether (100 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to

precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (100 mL). The filter-cake was dried under vacuum oven at 30°C to yield 12.0 g (81.0% yield) of the title compound as an off-white solid. 1H NMR (CDC1 ₃ , 400 MHz): δ 12.48 (broad s,-N ⁺ Me ₂ H, 1H), 6.80-6.88 (m , 2H), 4.20 (t, J= 6.4 Hz, 2H), 3.82 (s, 3H), 2.98 (m, 2H), 2.81 (s, 3H), 2.80 (s, 3H), 1.90 (m, 2H), 1.71 (m, 2H), 1.44 (m, 4H). MS (ESI): m/z 259.17 (M+H) ⁺ . Melting point: 1 16.7°C. Example 11

2-(diethylamino)ethyl methyl (2E)but-2-ene-l ,4-dioate (13)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 0.05 mol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (10 g, 0.05 mol) in 50 mL of dichloromethane (DCM) at ca. 0°C. 2-(diethylamino) ethanol (5.85 g, 0.05 mol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 6.02 g (52.57% yield) of the title compound was isolated as a viscous-oil.

Example 12

2-(diethylamino)ethyl methyl (2E)but-2-ene-l,4-dioate HC1 (14)

Following general Procedure B, 2-(dimethylamino)ethyl methyl (2E)but-2-ene-l ,4-dioate (5.5 g, 24 mmol) was dissolved in methyl tert-buty\ ether (50 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (50 mL). The filter-cake was dried under vacuum oven at 30°C to yield 4.0 g (62.8% yield) of the title compound as an off-white solid.

1H NMR (CDC1 ₃ , 400 MHz): δ 12.76 (broad s, -N ⁺ Et ₂ H, 1H), 6.93 (d, J= 16 Hz, 1H), 6.85 (d, J= 16 Hz, 1H), 4.77 (t, J= 4.8 Hz, 2H), 3.83 (s, 3H), 3.39 (m, 2H), 3.22 (m, 4H), 1.44 (t, J= 6.8 Hz, 6H). MS (ESI): m z 231.13 (M+H) ⁺ . Melting point: 137.9°C. Example 13

3-(diethylamino ropyl methyl (2E)but-2-ene-l,4-dioate (15)

Following general Procedure A, methyl hydrogen fumarate (MHF) (13g, 0.1 mol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (20 g, 0.1 mol) in 100 mL of dichloromethane (DCM) at ca. 0°C. 3-(diethylamino) propanol (1 1.6 g, 0.1 mol) and 4-N,N-dimethylaminopyridine (DMAP) (1 g, 0.008 mol) were added to the activated carboxylic acid. After synthesis, 13.2 g (54.3 % yield) of the title compound was isolated as a viscous-oil.

Example 14

3-(diethylamino ropyl methyl (2E)but-2-ene-l,4-dioate HCI

Following general Procedure B, 3-(diethylamino)propyl methyl (2E)but-2-ene-l,4-dioate (12.5 g, 51 mmol) was dissolved in methyl tert-butyl ether (100 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (100 mL). The filter-cake was dried under vacuum oven at 30°C to yield 10.2 g (71.0 % yield) of the title compound as an off-white solid.

1H NMR (CDC1 ₃ , 400 MHz): δ 12.37 (broad s, -N ⁺ Et ₂ H, 1H), 6.82-6.90 (m, 2H), 4.33 (t, J= 5.6 Hz, 2H), 3.83 (s, 3H), 3.08-3.18 (m, 6H), 2.34 (m, 2H), 1.43 (t, J= 6.8 Hz, 6H). MS (ESI): m/z 245.15 (M+H) ⁺ . Melting point: 153.2°C. Example 15

4-(diethylamino)butyl methyl (2E)but-2-ene-l,4-dioate (17)

Following general Procedure A, methyl hydrogen fumarate (MHF) (2.6 g, 20 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (4.0 g, 20 mmol) in 20 mL of dichloromethane (DCM) at ca. 0°C. 4-(diethylamino) butanol (2.9 g, 20 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (100 mg) were added to the activated carboxylic acid. After synthesis, 2.25 g (43.7% yield) of the title compound was isolated as a viscous-oil.

Example 16

4-(diethylamino)butyl methyl (2E)but-2-ene- 1 ,4-dioate HC1

Following general Procedure B, 4-(diethylamino)butyl methyl (2E)but-2-ene-l ,4-dioate (2.0 g, 7 mmol) was dissolved in methyl tert-butyl ether (10 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (10 mL). The filter-cake was dried under vacuum oven at 30°C to yield 1.1 g (48.2% yield) of the title compound as an off-white solid.

1H NMR (CDC1 ₃ , 400 MHz): δ 12.37 (broad s, -N ⁺ Et ₂ H, 1H), 6.82-6.90 (m, 2H), 4.33 (t, J= 6.4Hz, 2H), 3.82 (s, 3H), 3.08-3.18 (m, 4H), 3.00-3.05 (m, 2H), 1.94-1.99 (m, 2H), 1.77-1.86 (m, 2H), 1.42 (t, J= 7.2Hz, 6H). MS (ESI): m/z 259.17 (M+H) ⁺ . Melting point: 135.2°C. Example 17

5-(dieth lamino)pentyl methyl (2E)but-2-ene-l,4-dioate (19)

Following general Procedure A, methyl hydrogen fumarate (MHF) (1.3g, 10 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (2.0 g, 10 mmol) in 10 mL of dichloromethane (DCM) at ca. 0°C. 5-(diethylamino) pentanol (1.59 g, 10 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (100 mg) were added to the activated carboxylic acid. After synthesis, 1.23 g (45.3% yield) of the title compound was isolated as a viscous-oil.

Example 18

5-(diethylamino entyl methyl (2E)but-2-ene- 1 ,4-dioate HC1

Following general Procedure B, 5-(dimethylamino)pentyl methyl (2E)but-2-ene-l,4- dioate (1.2 g, 4.4 mmol) was dissolved in methyl tert-butyl ether (10 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to

precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (10 mL). The filter-cake was dried under vacuum oven at 30°C to yield 550 mg (40.4% yield) of the title compound as an off-white solid.

1H NMR (CDC1 ₃ , 400 MHz): δ 12.18 (broad s, -N ⁺ Et ₂ H, 1H), 6.82-6.90 (m, 2H), 4.22 (t, J= 6.4Hz, 2H), 3.82 (s, 3H), 3.07-3.15 (m, 4H), 2.92-3.00 (m, 2H), 1.86-1.95 (m, 2H), 1.72-1.80 (m, 2H), 1.42 (t, J= 7.2Hz, 6H), 1.41 (m, 2H). MS (ESI): m/z 273.18 (M+H) ⁺ .

Melting point: 96.0°C. Example 19

6-(diethylamino)hexyl methyl (2E)but-2-ene-l,4-dioate (21)

Following general Procedure A, methyl hydrogen fumarate (MHF) (1.3g, 10 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (2.0 g, 10 mmol) in 10 mL of dichloromethane (DCM) at ca. 0°C. 6-(diethylamino) hexanol (1.73 g, 10 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (100 mg) were added to the activated carboxylic acid. After synthesis, 1.35 g (47.3% yield) of the title compound was isolated as a viscous-oil.

Example 20

6-(diethylamino)hexyl methyl (2E)but-2-ene-l,4-dioate HC1 (22)

Following general Procedure B, 6-(dimethylamino)hexyl methyl (2E)but-2-ene-l,4- dioate (1.0 g, 3.5 mmol) was dissolved in methyl tert-buty\ ether (10 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to

precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (10 mL). The filter-cake was dried under vacuum oven at 30°C to 500 mg (45.4% yield) of the title compound as off- white solid.

1H NMR (CDC1 ₃ , 400 MHz): δ 11.93 (broad s, -N ⁺ Et ₂ H, 1H), 6.82-6.90 (m, 2H), 4.19 (t, J= 6.4Hz, 2H), 3.82 (s, 3H), 3.07-3.15 (m, 4H), 2.92-3.00 (m, 2H), 1.80-1.90 (m, 2H), 1.68-1.76 (m, 4H), 1.41 (t, J= 7.2Hz, 6H), 1.41(m,4 H). MS (ESI): m z 287.20 (M ⁺ H) ⁺ .

Melting point: N/A: hygroscopic salt. Example 21

Methyl (2-oxopyrrolidinyl)methyl (2E)but-2-ene-l,4-dioate (23)

Following general Procedure A, methyl hydrogen fumarate (MHF) (520 mg, 4 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (1.0 g, 5.2 mmol) in 10 mL of dichloromethane (DCM) at ca. 0°C. 1 -(hydroxymethyl)pyrrolidin-2- one (460 mg, 4 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (10 mg) were added to the activated carboxylic acid. After synthesis, 320 mg (35.2% yield) of the title compound was isolated as a viscous-oil.

1H NMR (CDC1 ₃ , 400 MHz): 5 6.90 (d, J=15.6Hz, 1H), 6.84 (d, J=15.6Hz, 1H), 5.46 (s, 2H), 3.82 (s, 3H), 3.56 (t, J= 7.2 Hz, 2H), 2.44 (t, J= 8.0 Hz, 2H), 2.07 (m, 2H). MS (ESI): m/z 287.20 (M+H)+.

Example 22

Methyl 2-(2-oxo rrolidinyl)ethyl (2E)but-2-ene-l,4-dioate (24)

Following general Procedure A, methyl hydrogen fumarate (MHF) (1.29 g, 10 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (2.5 g, 13.0 mmol) in 10 mL of dichloromethane (DCM) at ca. 0°C. N-(2-hydroxyethyl)-2- Pyrrolidinone (1.29 g, 10 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (50 mg) were added to the activated carboxylic acid. After synthesis, 900 mg (37.3% yield) of the title compound was isolated as off-white solid.

1H NMR (CDCI ₃ , 400 MHz): δ 6.82-6.90 (m, 2H), 4.35 (t, J= 6.0Hz, 2H), 3.82 (s, 3H), 3.60 (t, J= 5.6 Hz, 2H), 3.47 (t, J= 7.2Hz, 2H), 2.38 (t, J= 6.0Hz, 2H), 2.38 (m, 2H).

MS (ESI): m z 242.09 (M+H)+. Melting point: 45.8°C. Example 23

Methyl 3-(2-oxopyrrolidinyl)propyl (2E)but-2-ene-l ,4-dioate (25)

Following general Procedure A, methyl hydrogen fumarate (MHF) (2.6 g, 20 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (5 g, 26.1 mmol) in 20 mL of dichloromethane (DCM) at ca. 0°C. l-(3-Hydroxypropyl)pyrrolidin-2-one (3.14 g, 20 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (100 mg) were added to the activated carboxylic acid. After synthesis, 1.5 g (29.4% yield) of the title compound was isolated as viscous-oil.

1H NMR (CDC1 ₃ , 400 MHz): δ 6.82-6.90 (m, 2H), 4.22 (t, J= 6.2Hz, 2H), 3.81 (s, 3H), 3.40 (t, J= 6.8 Hz, 2H), 3.38 (t, J= 7.2Hz, 2H), 2.39 (t, J= 7.6Hz, 2H), 2.04 (m, 2H), 1.94 (m, 2H). MS (ESI): m/z 256.11 (M+H)+.

Example 24

2- {4-[(tert-butyl)oxycarbonyl]piperazinyl}ethyl methyl (2E)but-2-ene-l ,4-dioate (26)

Following general Procedure A, methyl hydrogen fumarate (MHF) (2.6 g, 20 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (5 g, 26.1 mmol) in 20 mL of dichloromethane (DCM) at ca. 0°C. l-Boc-4-(2-hydroxyethyl)piperazine (4.6 g, 20 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (50 mg) were added to the activated carboxylic acid. After synthesis, 4.3 g (63.23% yield) of the title compound was isolated as viscous-oil.

Example 25

Methyl 2-piperazinylethyl (2E)but-2-ene-l ,4-dioate dihydrochloride (27)

Following general Procedure B, 2-{4-[(tert-butyl)oxycarbonyl]piperazinyl}ethyl methyl (2E)but-2-ene-l ,4-dioate (4.3 g, 12.5 mmol) was dissolved in methyl tert-buty\ ether (10 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (10 mL). The filter-cake was dried under vacuum oven at 30°C to yield 2.6 g (41.4% yield) of the title compound as an off-white solid.

1H NMR (D ₂ 0, 400 MHz): δ 6.82 (d, J=16 Hz, 1H), 6.76 (d, J=16Hz, 1H), 4.65 (m, 2H), 4.50 (m, 2H), 3.68 (s, 3H), 3.62-3.45 (m, 8H). MS (ESI): m z 243.12 (M+H)+. Melting point: 96.5°C

Example 26

3-{4-[(tert-butyl)oxycarbonyl]piperazinyl}propyl methyl (2E)but-2-ene-l,4-dioate (28)

Following general Procedure A, methyl hydrogen fumarate (MHF) (2.6 g, 20 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (5 g, 26.1 mmol) in 20 mL of dichloromethane (DCM) at ca. 0°C. l-Boc-4-(3-hydroxypropyl)piperazine (4.8 g, 20 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (100 mg) were added to the activated carboxylic acid. After synthesis, 4.6 g (64.6% yield) of the title compound was isolated as viscous-oil.

Example 27

Methyl 3-piperazinylpropyl (2E)but-2-ene-l ,4-dioate dihyrochloride (29)

Following general Procedure B, 3-{4-[(tert-butyl)oxycarbonyl]piperazinyl}propyl methyl (2E)but-2-ene-l ,4-dioate (4.6 g, 12.9 mmol) was dissolved in methyl tert-buty\ ether (10 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (10 mL). The filter-cake was dried under vacuum oven at 30°C to yield 2.5 g (59.5% yield) of the title compound as an off-white solid.

1H NMR (CD ₃ OD, 400 MHz): 5 6.90 (d, J=16 Hz, IH), 6.85 (d, J=16Hz, IH), 4.33 (t, J= 6.4 Hz, 2H), 3.83 (s, 3H), 3.80-3.60 (m, 8H), 3.35 (m, 2H), 2.28 (m, 2H). MS (ESI): m/z 257.14

(M+H)+. Melting point: 196.8°C

Example 28

Methyl phenyl (2E)but-2-ene- 1 ,4-dioate (30)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (10 g, 50 mmol) in 20 mL of dichloromethane (DCM) at ca. 0°C. Phenol (4.8 g, 50 mmol) and 4-N,N- dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 5.7 g (55.3% yield) of the title compound was isolated as off-white solid.

1H NMR (CDCI3, 400 MHz): δ 7.40 (m, 2H), 7.26 (m, IH), 7.14 (m, 2H), 6.95 (d, J=15.6Hz, IH), 6.90 (d, J=15.6Hz, IH), 3.85 (s, 3H). MS (ESI): m/z 207.05 (M+H)+. Melting point: 58.2°C

Example 29

4-chlorophenyl methyl (2E)but-2-ene-l,4-dioate (31)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (10 g, 52.3 mmol) in 20 mL of dichloromethane (DCM) at ca. 0°C. 4-chlorophenol (6.4 g, 50 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 6.8 g (56.6% yield) of the title compound was isolated as off-white solid. 1H NMR (CDC1 ₃ , 400 MHz): δ 7.36 (m, 2H), 7.08 (m, 2H), 7.05 (d, J=15.6Hz, 1H), 7.04 (d, J=15.6Hz, 1H), 3.85 (s, 3H). MS (ESI): m z 241.01 , 243.01 (M+H)+ and +2 (isotope). Melting point: 47.1°C

Example 30

Methyl 4-meth lphenyl (2E)but-2-ene-l ,4-dioate (32)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (10 g,

52.3 mmol) in 20 mL of dichloromethane (DCM) at ca. 0°C. p-Cresol (5.4 g, 50 mmol) and 4-

Ν,Ν-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid.

After synthesis, 6.1 g (55.4% yield) of the title compound was isolated as a white solid.

1H NMR (CDCI ₃ , 400 MHz): δ 7.20 (m, 2H), 7.04 (m, 2H), 7.02 (m, 2H), 3.85 (s, 3H), 2.36 (s, 3H). MS (ESI): m z 221.07 (M+H)+. Melting point: 67.0°C

Example 31

Methyl 4-nitrophenyl (2E)but-2-ene-l,4-dioate (33)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (10 g, 52.3 mmol) in 20 mL of dichloromethane (DCM) at ca. 0°C. 4-nitrophenol (6.95 g, 50 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 6.2 g (49.40% yield) of the title compound was isolated as a white solid. 1H NMR (CDC1 ₃ , 400 MHz): δ 8.31 (m, 2H), 7.37 (m, 2H), 7.10 (d, J=15.6Hz, 1H), 7.06 (d, J=15.6Hz, 1H), 3.87 (s, 3H). MS (ESI): m z 252.04 (M+H)+. Melting point: 122.7°C.

Example 32

2,6-bis(methylethyl henyl methyl (2E)but-2-ene-l,4-dioate (34)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (10 g, 52.3 mmol) in 20 mL of dichloromethane (DCM) at ca. 0°C. 2,6-diisopropyl-phenol (Propofol, 8.9 g, 50 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 5.7 g (39.3% yield) of the title compound was isolated as a white solid.

1H NMR (CDCI ₃ , 400 MHz): δ 7.25 (m, 2H), 7.20 (m, 1H), 7.16 (d, J=15.6Hz, 1H), 7.10 (d, J=15.6Hz, 1H), 3.87 (s, 3H), 2.87 (m, 2H), 1.19 (d, J=3.2 Hz, 6H), 1.18 (d, J=3.2 Hz, 6H). MS (ESI): m/z 291.15 (M+H)+. Melting point: 66.5°C

Example 33

Methyl piperidyl (2E)but-2-ene-l ,4-dioate (35)

Following general Procedure A, methyl hydrogen fumarate (MHF) (1.30 g, 10 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (3 g, 15.7 mmol) in 50 mL of dichloromethane (DCM) at ca. 0°C. 1 -Hydro xypiperi dine (1.01 g, 10 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (50 mg) were added to the activated carboxylic acid. After synthesis, 1.84 g (86.3% yield) of the title compound was isolated as a viscous-oil.

Example 34

Methyl piperid l (2E)but-2-ene-l,4-dioate HC1

Following general Procedure B, Methyl piperidyl (2E)but-2-ene-l ,4-dioate (1.75 g, 8.2 mmol) was dissolved in methyl tert-butyl ether (10 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to precipitate/crystallize out as off- white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (10 mL). The filter-cake was dried under vacuum oven at 30°C to yield 1.05 g (51.4% yield) of the title compound as an off-white solid.

1H NMR (D ₂ 0, 400 MHz): δ 6.80 (d, J=15.6Hz, IH), 6.72 (d, J=15.6Hz, IH), 3.66 (s, 3H), 3.30 (m, 2H), 2.83 (m, 2H), 1.8-1.40 (m, 6H). MS (ESI): m/z 214.10 (M+H)+. Melting point: 109.0°C

Example 35

Diethylamino methyl (2E)but-2-ene-l,4-dioate (37)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (12 g, 62.8 mmol) in 50 mL of dichloromethane (DCM) at ca. 0°C. N,N-Diethyl hydroxylamine (4.45 g, 50 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 2.9 g (29.0% yield) of the title compound was isolated as a viscous-oil.

Example 36

Diethylamino meth l (2E)but-2-ene-l ,4-dioate HC1

Following general Procedure B, Diethylamino methyl (2E)but-2-ene-l ,4-dioate (2.9 g, 14.4 mmol) was dissolved in methyl tert-butyl ether (25 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (10 mL). The filter-cake was dried under vacuum oven at 30°C to yield 3.0 g (85.3% yield) of the title compound as an off-white solid.

1H NMR (CDC1 ₃ , 400 MHz): δ 6.95 (d, J=15.6Hz, 1H), 6.90 (d, J=15.6Hz, 1H), 3.82 (s, 3H), 2.98 (q, J= 6.8 Hz, 4H), 1.13 (t, J= 6.8 Hz, 6H). MS (ESI): m/z 202.10 (M+H)+. Melting point: hygroscopic solid

Example 37

Dimethylamino methyl (2E)but-2-ene-l,4-dioate (39)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (12 g, 62.8 mmol) in 50 mL of dichloromethane (DCM) at ca. 0°C. N,N-Dimethyl hydroxylamine (3.05 g, 50 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 2.0 g (23.2% yield) of the title compound was isolated as a viscous-oil.

Example 38

Dimethylamino dioate HC1

Following general Procedure B, Dimethylamino methyl (2E)but-2-ene-l,4-dioate (2.0 g, 11.5 mmol) was dissolved in methyl tert-butyl ether (25 mL). The resulting clear reaction mixture is cooled to 0°C (ice bath). 1.1 equivalent of hydrochloride in dioxane (4M) was slowly added over a period of 30 minutes. During this period the product starts to precipitate/crystallize out as off-white solid. The solid product was separated by filtration and the filter-cake was washed with methyl tert-butyl ether (10 mL). The filter-cake was dried under vacuum oven at 30°C to yield 2.0 g (95.6% yield) of the title compound as an off-white solid.

1H NMR (CDC1 ₃ , 400 MHz): δ 6.90 (d, J=15.6Hz, IH), 6.82 (d, J=15.6Hz, IH), 3.81 (s, 3H),

2.82 (s, 6H). MS (ESI): m/z 174.06 (M+H)+. Melting point: 101.1°C

Example 39

Cyclohexyl meth l (2E)but-2-ene-l,4-dioate (41) Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (12 g, 62.8 mmol) in 50 mL of dichloromethane (DCM) at ca. 0°C. Cyclohexanol (5.0 g, 50 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 5.9 g (55.6% yield) of the title compound was isolated as viscous-oil.

'H NMR (CDCL3, 400 MHz): δ 6.90-6.82 (m, 2H), 4.88 (m, 1H), 3.80 (s, 3H), 1.86 (m, 2H), 1.76 (m, 2H), 1.66-1.30 (m, 6H). MS (ESI): m/z 213.10 (M+H)+.

Example 40

Cyclopentyl meth l (2E)but-2-ene- 1 ,4-dioate (42)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (12 g, 62.8 mmol) in 50 mL of dichloromethane (DCM) at ca. 0°C. Cyclopentanol (4.3 g, 50 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 5.9 g (59.6% yield) of the title compound was isolated as a viscous-oil. 'H NMR (CDCI3, 400 MHz): δ 6.90-6.82 (m, 2H), 5.27 (m, 1H), 3.80 (s, 3H), 1.92-1.60 (m, 8H). MS (ESI): m z 199.08 (M+H)+. Example 41

2H-3,4,5,6-tetrahydro ran-2-yl methyl (2E)but-2-ene- 1 ,4-dioate (43)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (12 g, 62.8 mmol) in 50 mL of dichloromethane (DCM) at ca. 0°C. 2-Hydroxytetrahydropyran (5.1 g, 50 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 5.9 g (55.1% yield) of the title compound was isolated as a viscous-oil.

1H NMR (CDC1 ₃ , 400 MHz): δ 6.92-6.84 (m, 2H), 6.11 (m, 1H), 3.90 (m, 1H), 3.80 (s, 3H), 3.73 (m, 1H), 1.90-1.50 (m, 6H). MS (ESI): m/z 215.08 (M+H)+.

Example 42

(Methoxycarbonyl)methyl methyl (2E)but-2-ene-l,4-dioate (44)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (12 g, 62.8 mmol) in 50 mL of dichloromethane (DCM) at ca. 0°C. Methyl glycolate (4.5 g, 50 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 6.7 g (66.3% yield) of the title compound was isolated as a white solid. 1H NMR (CDCI3, 400 MHz): δ 6.96-6.92 (m, 2H), 4.74 (s, 2H), 3.80 (s, 3H), 3.78 (s, 3H). MS (ESI): m/z 203.04 (M+H)+. Melting point: 51.0°C

Example 43

(ethoxycarbonyl)methyl methyl (2E)but-2-ene-l,4-dioate (45)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (12 g, 62.8 mmol) in 50 mL of dichloromethane (DCM) at ca. 0°C. Ethyl glycolate (4.5 g, 50 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 5.3 g (49.0% yield) of the title compound was isolated as a white solid. 1H NMR (CDCI ₃ , 400 MHz): δ 6.96-6.92 (m, 2H), 4.73 (s, 2H), 4.25 (q, J= 7.2 Hz, 2H),

3.82 (s, 3H), 1.29 (t, J= 7.2 Hz, 3H). MS (ESI): m z 217.06 (M+H)+. Melting point: 44.8°C Example 44

Methyl [benz loxycarbonyljmethyl (2E)but-2-ene-l,4-dioate (46)

Following general Procedure A, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (12 g, 62.8 mmol) in 50 mL of dichloromethane (DCM) at ca. 0°C. Benzyl glycolate (6.5 g, 50 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 7.2 g (51.8% yield) of the title compound was isolated as a white solid. 1H NMR (CDC1 ₃ , 400 MHz): δ 7.34 (m, 5H), 6.96-6.92 (m, 2H), 5.51 (s, 2H), 4.77 (s, 2H), 3.82 (s, 3H). MS (ESI): m/z 279.07 (M+H)+. Melting point: 63.8°C

Example 45

(Methoxy-N-methylcarbonylamino)methyl methyl (2E)but-2-ene- 1 ,4-dioate (47)

Following general Procedure C, methyl hydrogen fumarate (MHF) (3.9 g, 30 mmol) and triethylamine (3.5 g, 34.6 mmol) were added in 10 mL of tetrahydrofuran (THF) at ca. 0°C. N- methyl-N-methyloxycarbonylaminomethyl chloride (4.11 g, 30 mmol) was added to the carboxylic acid salt. Then the reaction mixture was warmed to 50°C for 18 h. After synthesis, 2.5 g (36.2% yield) of the title compound was isolated as a viscous-oil.

1H NMR (CDCI ₃ , 400 MHz): 6.96-6.94 (m, 2H), 5.50 (br s, 2H), 3.81 (s, 3H), 3.76 (s, 3H), 3.03 (s, 3H). MS (ESI): m/z 232.07 (M+H)+.

Example 46

(Ethoxy-N-methylcarbonylamino)methyl methyl (2E)but-2-ene- 1 ,4-dioate (48)

Following general Procedure C, methyl hydrogen fumarate (MHF) (6.5 g, 50 mmol) and triethylamine (6.0 g, 59.4 mmol) were added in 50 mL of tetrahydrofuran (THF) at ca. 0°C. N- methyl-N-ethyloxycarbonylaminomethyl chloride (7.5 g, 50 mmol) was added to the carboxylic acid salt. Then the reaction mixture was warmed to 50°C for 18 h. After synthesis, 6.0 g (49.0% yield) of the title compound was isolated as viscous-oil.

1H NMR (CDC1 ₃ , 400 MHz): δ 6.90-6.86 (m, 2H), 4.49 (br s, 2H), 4.20 (q, J= 6.8 Hz, 2H), 3.82 (s, 3H), 3.03 (s, 3H), 1.29 (t, J= 6.8 Hz, 3H). MS (ESI): m/z 246.08 (M+H)+. Example 47

Methyl [N-methyl(phenylmethoxy)carbonylamino]methyl (2E)but-2-ene-l,4-dioate (49)

Following general Procedure C, methyl hydrogen fumarate (MHF) (3.9 g, 30 mmol) and triethylamine (3.5 g, 34.6 mmol) were added in 30 mL of tetrahydrofuran (THF) at ca. 0°C. N- methyl-N-benzyloxycarbonylaminomethyl chloride (6.3 g, 30 mmol) was added to the carboxylic acid salt. Then the reaction mixture was warmed to 50°C for 18 h. After synthesis, 5.6 g (62.2% yield) of the title compound was isolated as a viscous-oil.

1H MR (CDCI ₃ , 400 MHz): 7.36 (m, 5H), 6.90-6.82 (m, 2H), 5.51 (br s, 2H), 5.19 (s, 2H), 3.82 (s, 3H), 3.05 (s, 3H). MS (ESI): m/z 308.10 (M+H)+.

Example 48

Methyl (2E)-3-{N-[(lR)-l-(ethoxycarbonyl)-2-methylpropyl]carbamoyl} prop-2-enoate (50) Following general Procedure A, methyl hydrogen fumarate (MHF) (1.30 g, 10 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (2.5 g, 13 mmol), and triethylamine (1.01 g, 10 mmol) in 10 mL of dichloro methane (DCM) at ca. 0°C. D-Valine, ethyl ester.HCl (1.81 g, 10 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (500 mg) were added to the activated carboxylic acid. After synthesis, 1.2 g (46.7% yield) of the title compound was isolated as a viscous-oil.

^! H NMR (CDC1 ₃ , 400 MHz): δ 7.0 (d, J=15.6Hz, 1H), 6.84 (d, J=15.6Hz, 1H), 6.44 (d, J=8.0 Hz, -NH, 1H), 4.65 (dd, J=15.6Hz, 1H), 4.22 (m, 2H), 3.81 (s, 3H), 2.22 (m, 1H), 1.30 (t, J=7.2Hz, 3H), 0.96 (d, J=7.2 Hz, 3H), 0.94 (d, J=7.2Hz, 3H). MS (ESI): m z 258.12 (M+H)+.

Example 49

(2,5-Dioxoazolidinyl)methyl (2E)but-2-ene-l,4-dioate

Following general Procedure A, methyl hydrogen fumarate (MHF) (1.30 g, 10 mmol) was activated with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ED AC) (2.5 g, 13 mmol) in 10 mL of dichloromethane (DCM) at ca. 0°C. N-(Hydroxymethyl)succinimide (1.29 g, 10 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (50 mg) were added to the activated carboxylic acid. After synthesis, 800 mg (33.3% yield) of the title compound was isolated as off-white solid. Melting point: 120.4°C.

1H NMR (CDC13, 400 MHz): δ 6.88 (d, J=15.6Hz, 1H), 6.80 (d, J=15.6Hz, 1H), 5.63 (s, 2H), 3.80 (s, 3H), 2.81 (s, 2H). MS (ESI): m/z 242.19 (M+H)+.

[0092] The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions and methods provided herein and are not to be construed in any way as limiting their scope. In the examples, all temperatures are in degrees Celsius (unless otherwise indicated). Compounds that can be prepared in accordance with the methods provided herein along with their biological and PK data are presented in following Tables. The syntheses of these representative compounds are carried out in accordance with the methods set forth above.

Exemplary Compounds provided herein

The following compounds, and pharmaceutically acceptable salts thereof, have been or can be prepared according to the synthetic methods described herein.

TABLE 1: Exemplary Compounds of the Disclosure

105

Description 1

Methods for Determining Stability of Monomethyl and monoethyl fumarate Prodrugs in

Vitro

Certain monomethyl and monoethyl fumarate prodrugs of the disclosure may or may not themselves be pharmacologically active, and are metabolized in vivo to produce a

pharmacologically active metabolite. For a prodrug, it can be desirable that the prodrug remains intact (i.e., uncleaved) while in the systemic circulation and be cleaved (i.e., to release the parent drug) in the target tissue. Alternatively, it can be desirable that the prodrug remains intact (i.e., uncleaved) while in the gastrointestinal tract and be cleaved (i.e., to release the parent drug) after being absorbed or taken up from the gastrointestinal lumen, e.g. , in either the enterocytes lining the gastrointestinal lumen or in the blood. For pharmacologically active compounds, it can be desirable that the compound remains intact in the gastrointestinal tract.

A useful level of stability can at least in part be determined by the mechanism and pharmacokinetics of the prodrug or active compound. In general, prodrugs or active compounds that are more stable in pancreatin or colonic wash assay, and are more labile in rat plasma, human plasma, rat liver S9, and/or human liver S9 preparations, can be useful as an orally administered prodrug or active compound. In general, prodrugs or active compounds that are more stable in rat plasma, human plasma, rat liver S9, and/or human liver S9 preparations, and which are more labile in cell homogenate preparations such CaCo2 S9 preparations, can be useful as systemically administered prodrugs or active compounds and/or can be more effective in delivering a prodrug or active compound to a target tissue. In general, prodrugs or active compounds that are more stable in a range of pH physiological buffers (pH 6.0 to pH 8.5) can be more useful as orally administered prodrugs or active compounds. In general, prodrugs or active compounds that are more labile in cell homogenate preparations, such CaCo2 S9 preparations, can be intracellularly metabolized. The results of tests, such as those described in this example, for determining the enzymatic or chemical cleavage of compounds in vitro can be used to select prodrugs for in vivo testing.

The stabilities of prodrugs or active compounds can be evaluated in one or more in vitro systems using a variety of preparations following methods known in the art. For example, methods used to determine the stability of prodrugs in Caco2 S9 homogenate, rat liver S9, rat plasma, porcine pancreatin, rat colonic wash, and pH 8.0 buffer are described herein.

CaCo2 S9 homogenate is prepared using the following procedure. CaCo2 cells are grown in culture for 21 days prior to harvesting. Culture medium is removed from the culture vessel and the monolayer is rinsed twice with 10-15 mL chilled phosphate buffered saline (PBS) buffer. PBS buffer (7-10 mL) is added to the flask and the cells scraped from the growth surface and transferred to a centrifuge tube. The cells are pelleted by centrifugation at 1,500 rpm for 5 min at 4°C. The supernatant is removed and the cell pellet washed with ice cold PBS and re -pelleted by centrifugation. The supernatant is removed and the pellet re-suspended in cell lysis buffer (0.15M KC1 and 10 mM sodium phosphate buffer, pH 7.4). Cells are lysed by sonication at 4°C using a probe sonicator. The lysed cells are then transferred to vials and centrifuged at 1,600 rpm for 10 min at 4°C to remove intact cells, nuclei, and large cellular debris. The supernatant is removed and transferred to a tube for centrifugation at 8,600 rpm for 20 min at 4°C. After centrifugation, the resulting supernatant, representing the CaCo2 cell homogenate S9 fraction, is carefully removed and aliquoted into vials for storage at -80°C until the time of use. At the time of use, CaCo2 S9 lysate is diluted to 0.5 mg/mL in 0.1M Tris buffer, pH 7.4.

Rat liver S9 (XenoTech, Lenexa, KS; R1000.S9, 20 mg/mL) is diluted to 0.5 mg/mL in 0.1 M potassium phosphate buffer at pH 7.4 and ImM NADPH cofactor.

Rat plasma (Pel-Freez ^® Biologicals, Rogers, AR; 36150) is used as obtained from the supplier. Porcine pancreatin (Sigma Aldrich, St. Louis, MO; P1625-100G) is diluted to 10 mg/mL in O. lM Tris buffer, pH 7.4.

To prepare the rat colonic wash, the colon between the ceacum and rectum is resected from a euthanized rat. Five to 10 mL of PBS pH 7.4 buffer (depending on the weight of the rat) is flushed into the lumen of the large intestine and collected into a 250 mL glass beaker at 0°C (ice bath). The colonic wash is transferred into 10 mL conical tubes using a 10 mL syringe fitted with a filter. Samples of 0.5 mL colonic wash are stored at -80°C until the time of use. Colonic wash is used without dilution.

The enzymatic stability assays for a compound in CaCo2 S9, rat liver S9, rat plasma, pig pancreatin, and rat colonic wash are performed using the following procedure. Ninety (90) μL· of lysate is aliquoted to designated tubes on a cluster plate. The lysate is pre-incubated for 10 min at 37°C. With the exception of the t(0) time point, 10 of a 400 μΜ solution of test compound in 0.1M Tris buffer, pH 7.4 is added to multiple wells, representing different incubation times. The samples are incubated at 37°C. At each time point, the reaction is quenched by adding 300 μL· of 100% ethanol. The samples are thoroughly mixed, the tubes transferred to a V-bottom plate, and stored at -20°C. For the t(0) time point, the lysate is quenched with 300 μL· of ice cold 100% ethanol, thoroughly mixed, 10 μL· oΐ 400 μΜ test compound is added and mixed, and the sample tube transferred to a V-bottom plate and stored at -20°C. For analysis, 180 μL· from each sample is transferred to a 96 well V-bottom plate and sealed. After all time points are collected, the plate is centrifuged for 10 min at 5600 rpm at 4°C. One-hundred fifty (150) μL· from each well is then transferred to a 96 well round bottom plate. Samples are analyzed using LC/MS/MS to determine the concentrations of the compound and/or metabolite thereof.

For the pH 8.0 stability studies, 190 μΐ, of 150 mM NaH ₂ P0 ₄ buffer pH 8.0 is added to each sample tube. Ten (10) μL· of 20 mM test compound is added to each tube and mixed. The samples are incubated for 60 min at 37°C. Following incubation, the samples are transferred to room temperature and 800 μL· of 50% acetonitrile in water is added to each tube. Samples are analyzed using LC/MS/MS to determine the concentrations of the compound and/or metabolite thereof.

LC/MS/MS analysis for MHF is performed using an API 4000 equipped with an Agilent 1 100 HPLC and a Leap Technologies autosampler. An HPLC Phenomenex Onyx Monolithic CI 8 (CHO-7644) column at a temperature of 35°C, flow rate of 2.0 mL/min, injection volume of 30 \L, and a 3-min run time is used. The mobile phase AI is 0.1% formic acid in water and Mobile phase All is 0.1% formic acid in acetonitrile. The gradient is 98% AI /2% All at time 0; 98% AI / 2% All at time 0.1 min; 5% AI / 95% All at time 1.4 min; 5% AI / 95% All at time 2.2 min; 98% AI / 2% All at time 2.3 min; and 98% AI / 2% All at time 3.0 min. MHF content is determined using negative ion mode (Ql 128.94; Q2 71).

Description 2

Monomethyl Fumarate and Monoethyl fumarate Bioavailability Following Oral Administration of Monomethyl and monoethyl fumarate Prodrugs Rats are obtained commercially and are pre-cannulated in the jugular vein. Animals are conscious at the time of the experiment. All animals are fasted overnight and until 4 hours post- dosing of a compound of the disclosure.

Blood samples (0.3 mL/sample) are collected from all animals prior to dosing and at different time-points up to 24 h post-dose into tubes containing EDTA. Two aliquots (100 \iL each) are quenched with 300 \L methanol and stored at -20°C prior to analysis.

To prepare analysis standards, 90 \iL of rat blood is quenched with 300 \iL methanol followed by 10 \iL of spiking standard and/or 20 \iL of internal standard. The sample tubes are vortexed for at least 2 min and then centrifuged at 3,400 rpm for 20 min. The supernatant is then transferred to an injection vial or plate for analysis by LC-MS-MS.

To prepare samples for analysis, 20 \iL of internal standard is added to each quenched sample tube. The sample tubes are vortexed for at least 2 min and then centrifuged at 3,400 rpm for 20 min. The supernatant is then transferred to an injection vial or plate for analysis by LC/MS/MS.

LC/MS/MS analysis can be performed using an API 4000 (MS 12) equipped with Agilent 1100 HPLC and a Leap Technologies autosampler. The following HPLC column conditions are used: HPLC column: Onyx Monolithic CI 8 Phenomex (PN CHO-7644), 35°C; flow rate 2.0 mL/min; injection volume 30 μί; run time 3 min; mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile (ACN); gradient: 98%A / 2%B at 0.0 min;

98%A / 2%B at 0.1 min; 5%A / 95%B at 1.4 min; 5%A / 95%B at 2.2 min; 98%A / 2%B at 2.3 min; and 98%A / 2%B at 3.0 min. MHF is monitored in negative ion mode. Non-compartmental analysis is performed using WinNonlin software (v.3.1 Professional Version, Pharsight Corporation, Mountain View, California) on individual animal profiles.

Summary statistics on major parameter estimates is performed for C _max (peak observed concentration following dosing), T _max (time to maximum concentration is the time at which the peak concentration is observed), AUC ₍ o-t) (area under the plasma concentration-time curve from time zero to last collection time, estimated using the log-linear trapezoidal method), AUC _(0-∞) , (area under the plasma concentration time curve from time zero to infinity, estimated using the log-linear trapezoidal method to the last collection time with extrapolation to infinity), and ti/2, _z (terminal half-life).

A compound of the disclosure is administered by oral gavage to groups of four to six adult male Sprague-Dawley rats (about 250 g). Animals are conscious at the time of the experiment. A compound of the disclosure is orally or colonically administered in 3.4% Phosal at a dose of 70 mg- equivalents MHF per kg body weight.

The percent relative bioavailability (F%) of the administered compound or metabolite thereof is determined by comparing the area under the respective concentration vs time curve (AUC) following oral or colonic administration of a compound of the disclosure with the AUC of the concentration vs time curve following intravenous administration of the compound of the disclosure, respectively, on a dose-normalized basis.

The %F can be reported as the mean %F of all animals dosed orally with the compound of the disclosure at the specified level.

Description 3

Animal Model for Assessing Therapeutic Efficacy of Monomethyl and monoethyl fumarate

Prodrugs for Treating Multiple Sclerosis

Animals and Experimental Autoimmune Encephalomyelitis Induction

Female C57BL/6 mice, 8-10 weeks old (Harlan Laboratories, Livermore, CA), are immunized subcutaneously in the flanks and mid-scapular region with 200μg of myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35_5s) (synthesized by Invitrogen) emulsified (1 : 1 volume ratio) with complete Freund's adjuvant (CFA) (containing 4 mg/mL Mycobacterium tuberculosis) . Emulsion is prepared by the syringe-extrusion method with two glass Luer-Lock syringes connected by a 3-way stopcock. Mice are also given an intraperitoneal injection of 200 ng pertussis toxin (List Biological Laboratories, Inc, Campbell, CA) on the day of immunization and on day two post immunization. Mice are weighed and examined daily for clinical signs of experimental autoimmune encephalomyelitis (EAE). Food and water is provided ad libitum and once animals start to show disease, food is provided on the cage bottom. All experiments are approved by the Institutional Animal Care and Use Committee.

Clinical Evaluation

Mice are scored daily beginning on day 7 post immunization. The clinical scoring scale is as follows (Miller and Karplus, Current Protocols in Immunology 2007, 15.1.1-15.1.18): 0 = normal; 1 = limp tail or hind limb weakness (defined by foot slips between bars of cage top while walking); 2 = limp tail and hind limb weakness; 3 = partial hind limb paralysis (defined as no weight bearing on hind limbs but can still move one or both hind limbs to some extent); 4 = complete hind limb paralysis; 5 = moribund state (includes forelimb paralysis) or death.

Treatment

Compound(s) of the disclosure are dissolved in 0.5% methocellulose/0.1% Tween80 in distilled water and administered by oral gavage twice daily starting from day 3 post- immunization until termination. Dexamethasone is dissolved in IX PBS buffer and administered subcutaneously once daily. Treatment groups are, for example, as follows: vehicle alone, 15 mg/kg DMF, 20 mg/kg compound of the disclosure, and 1 mg/kg dexamethasone.

Alternate Animal Models of Multiple Sclerosis

The following experiment confirmed that MHF is the active moiety of both MHF prodrugs DMF and the compounds of the disclosure and examined the relationship between MHF exposure and effect in animal models of multiple sclerosis (MS). Efficacy of representative compound of the disclosure and DMF is compared in the MOG35-55 mouse EAE model of multiple sclerosis. C57BL/6 mice (6 females) are injected subcutaneously with MOG35-55 peptide in CFA with Mycobacterium tuberculosis. Pertussis toxin (200 mg) is injected IV on Day 0 and Day 2 post-immunization. Animals received oral test compound or DMF (90 mg-eq MHF/kg twice daily) or vehicle on Days 3 to 29. Daily EAE clinical disease scores (5 point scale) are recorded. End of study MHF blood levels are determined by LC/MS/MS.

Ill Description 4

Use of an Animal Model to Assess Efficacy in Treating Psoriasis

The severe, combined immunodeficient (SCID) mouse model can be used to evaluate the efficacy of compounds for treating psoriasis in humans (Boehncke, Ernst Schering Res Found Workshop 2005, 50, 213-34; and Bhagavathula et ah, J Pharmacol Expt'l Therapeutics 2008, 324(3), 938-947).

SCID mice are used as tissue recipients. One biopsy for each normal or psoriatic volunteer (human) is transplanted onto the dorsal surface of a recipient mouse. Treatment is initiated 1 to 2 weeks after transplantation. Animals with the human skin transplants are divided into treatment groups. Animals are treated twice daily for 14 days. At the end of treatment, animals are photographed and then euthanized. The transplanted human tissue along with the surrounding mouse skin is surgically removed and fixed in 10% formalin and samples obtained for microscopy. Epidermal thickness is measured. Tissue sections are stained with an antibody to the proliferation-associated antigen Ki-67 and with an anti-human CD3 ⁺ monoclonal antibody to detect human T lymphocytes in the transplanted tissue. Sections are also probed with antibodies to c-myc and β-catenin. A positive response to treatment is reflected by a reduction in the average epiderma thickness of the psoriatic skin transplants. A positive response is also associated with reduced expression of Ki-67 in keratinocytes. Alternate Animal Models of Multiple Sclerosis and Pson ^' asMmidquimod model of skin inflammation (Fits et al The Journal of Immunology, 2009, 182: 5836-5845). 10-12 week old BALB/c, I117c+/+ or 1117c-/-, or I117re+/+ or II 17re— /— mice were administered 50 mg Aldara cream (5% Imidquimod in Graceway, 3M) in the shaved back and right ear daily for 5 days. Clinical scoring and ear thickness measurements were performed daily. Scoring was based upon the manifestation of psoriatic symptoms, such as erythema, scaling and thickness: 0, No disease. 1, Very mild erythema with very mild thickening and scaling involving a small area. 2, Mild erythema with mild thickening and scaling involving a small area. 3, Moderate erythema with moderate thickening and scaling (irregular and patchy) involving a small area (<25%). 4, Severe erythema with marked thickening and scaling (irregular and patchy) involving a moderate area (25-50%). 5, Severe erythema with marked thickening and scaling (irregular and patchy) involving a large area (>50%). Ear and back tissue were harvested on day 5 for histological evaluation.

Efficacy of compounds of the disclosure and DMF is compared in the imiquimod (IMQ) mouse model of psoriasis. Balb/c mice (10 males/group) receive daily topical IMQ (5% cream) on shaved back and right ear for 5 days as described above. Animals receive oral dose of a representative compound of the disclosure or DMF (45 or 90 mg-eq MMF/kg twice daily) or vehicle from Day -5 to Day 5. Erythema score is the primary outcome measure.

Description 5

Animal Model for Assessing Therapeutic Efficacy of Monomethyl and monoethyl fumarate

Prodrugs for Treating Multiple Sclerosis

Experiments are conducted on female mice aged 4-6 weeks belong to the C57BL/6 strain weighing 17-20 g. Experimental autoimmune encephalomyelitis (EAE) is actively induced using >95% pure synthetic myelin oligodendrocyte glycoprotein peptide 35-55 (MOG ₃₅ _5 ₅ )

(synthesized by Invitrogen). Each mouse is anesthetized and receives 200 μg of MOG ₃₅ _55 peptide and 15 μg of Saponin extract from Quilija bark emulsified in 100 of phosphate- buffered saline. A 25 volume is injected subcutaneously over four flank areas. Mice are also intraperitoneally injected with 200 ng of pertussis toxin in 200 μL of PBS. A second, identical injection of pertussis toxin is given after 48 h.

A compound of the disclosure is administered at varying doses. Control animals receive 25 iL of DMSO. Daily treatment extends from day 26 to day 36 post-immunization. Clinical scores are obtained daily from day 0 post-immunization until day 60. Clinical signs are scored using the following protocol: 0, no detectable signs; 0.5, distal tail limpness, hunched appearance and quiet demeanor; 1, completely limp tail; 1.5, limp tail and hindlimb weakness (unsteady gait and poor grip with hind limbs); 2, unilateral partial hind limb paralysis; 2.5, bilateral hind limb paralysis; 3, complete bilateral hindlimb paralysis; 3.5, complete hindlimb paralysis and unilateral forelimb paralysis; 4, total paralysis of hind limbs and forelimbs (Eugster et al., Eur J Immunol 2001, 31, 2302-2312).

Inflammation and demyelination are assessed by histology on sections from the CNS of EAE mice. Mice are sacrificed after 30 or 60 days and whole spinal cords are removed and placed in 0.32 M sucrose solution at 4°C overnight. Tissues are prepared and sectioned. Luxol fast blue stain is used to observe areas of demyelination. Haematoxylin and eosin staining is used to highlight areas of inflammation by darkly staining the nuclei of mononuclear cells. Immune cells stained with H&E are counted in a blinded manner under a light microscope. Sections are separated into gray and white matter and each sector is counted manually before being combined to give a total for the section. T cells are immunolabeled with anti-CD3+ monoclonal antibody. After washing, sections are incubated with goat anti-rat HRP secondary antibody. Sections are then washed and counterstained with methyl green. Splenocytes isolated from mice at 30 and 60 days post-immunization are treated with lysis buffer to remove red blood cells. Cells are then re- suspended in PBS and counted. Cells at a density of about 3 x 10 ⁶ cells/mL are incubated overnight with 20 μg/mL of MOG peptide. Supernatants from stimulated cells are assayed for IFN-γ protein levels using an appropriate mouse IFN-γ immunoassay system.

Description 6

Use of an Animal Model to Assess Efficacy in Treating Inflammatory Bowel Disease

Animal models of inflammatory bowel disease are described by Jurjus et al., J

Pharmaocol Toxicol Methods 2004, 50, 81 -92; Villegas et al., Int Ί Immunopharmacol 2003, 3, 1731-1741 ; and Murakami et al., Biochemical Pharmacol 2003, 66, 1253-1261. For example, the following protocol can be used to assess the efficacy of a compound of the disclosure for treating inflammatory bowel disease.

Female ICR mice are used. Mice are divided into treatment groups. Groups are given either water (control), 5% DSS in tap water is given at the beginning of the experiment to induce colitis, or various concentrations of test compound. After administering test compound for 1 week, 5% DSS in tap water is also administered to the groups receiving test compound for 1 week. At the end of the experiment, all mice are sacrificed and the large intestine is removed. Colonic mucosa samples are obtained and homogenized. Proinflammatory mediators {e.g., IL- la, IL-Ι β, TNF-a, PGE2, and PGF2a) and protein concentrations are quantified. Each excised large intestine is histologically examined and the damage to the colon scored.

Description 7

Clinical Trial for Assessing Efficacy in Treating Asthma

Adult subjects (nonsmokers) with stable mild-to-moderate asthma are enrolled (see, e.g., Van Schoor and Pauwels, Eur Respir J 2002, 19, 997- 1002). A randomized, double-blind, placebo-controlled, two-period crossover design is used. On screening day 1 , patients undergo a methacholine challenge (< 8 mg/mL). The baseline forced expiratory volume in one second (FEVl) prior to each subsequent challenge must be within 15% of the screening baseline FEVl obtained at the first visit. A neurokinin challenge (l xlO ^-6 mol mL) on screening day 2 is performed 24-72 h later. Study-period one commences within 10 days after visit two. First, a methacholine and a neurokinin-A (NKA) challenge is performed on days 1 and 0, respectively. At visit four, test compound is administered at an appropriate dose and for an appropriate period of time. On the last 2 days of the treatment period, methacholine and NKA challenges are repeated. Following treatment-period one, there is a washout period of about 5 weeks, following which the patients crossed over to another medication or placebo in study period two, which is identical to period one. Pulmonary function tests are performed using a spirometer. The metacholine challenge is performed by inhaling doubling concentrations of methacholine until the FEVl falls by >20% of the post-diluent baseline FEVl of that day as described by Cockcroft et al., Clin Allergy 1977, 7, 235-243. NKA challenge is performed by inhaling increasing concentrations of NKA as described by Van Schoor et al., Eur Respir J 1998, 12, 17-23. The effect of a treatment on airway responsiveness is determined using appropriate statistical methods.

Description 8

Use of an Animal Model to Assess Efficacy in Treating Chronic Obstructive Pulmonary

Disease

An animal model using mice chronically exposed to cigarette smoke can be used for assessing efficacy in treating emphysema (see, e.g., Martorana et ah, Am J Respir Crit Care Med 2005, 172, 848-835; and Cavarra et al, Am J Respir Crit Care Med 2001 , 164, 886-890). Six- week old C57B1/6J male mice are used. In the acute study, the mice are exposed either to room air or to the smoke of five cigarettes for 20 minutes. In the chronic study, the mice are exposed to either room air or to the smoke of three cigarettes/day, for 5 days/week, for 7 months.

For the acute study, mice are divided into three groups of 40 animals each. These groups are then divided into four subgroups of 10 mice each as follows: (1) no treatment/air-exposed; (2) no treatment/smoke-exposed; (3) a first dose of test compound plus smoke-exposed; and (4) a second dose of test compound. In the first group, trolox equivalent antioxidant capacity is assessed at the end of the exposure in bronchoalveolar lavage fluid. In the second group, cytokines and chemokines are determined in bronchoalveolar lavage fluid using a commercial cytokine panel at 4 hours; and in the third group bronchoalveolar lavage fluid cell count is assessed at 24 hours.

For the chronic study, five groups of animals are used: (1) no treatment/air-exposed; (2) a first dose of a test compound plus air-exposed; (3) no treatment/smoke-exposed; (4) a second dose of the test compound plus smoke-exposed; and (5) the first dose of the test compound plus smoke exposed. Seven months after chronic exposure to room air or cigarette smoke, 5 to 12 animals from each group are sacrificed and the lungs fixed intratracheally with formalin. Lung volume is measured by water displacement. Lungs are stained. Assessment of emphysema includes mean linear intercept and internal surface area. The volume density of macrophages, marked immunohistochemically with anti-mouse Mac-3 monoclonal antibodies is determined by point counting. A mouse is considered to have goblet cell metaplasia when at least one or more midsize bronchi/lung showed a positive periodic acid-Schiff staining. For the determination of desmosine, fresh lungs are homogenized, processed, and analyzed by high-pressure liquid chromato graphy .

Description 9

Animal Models for Assessing Therapeutic Efficacy of Monomethyl and monoethyl fumarate Prodrugs for Treating Parkinson's Disease

MPTP Induced Neurotoxicity

MPTP, or l-methyl-4-phenyl-l ,2,3,6-tetrahydropyridine is a neurotoxin that produces a Parkinsonian syndrome in both humans and experimental animals. Studies of the mechanism of MPTP neurotoxicity show that it involves the generation of a major metabolite, MPP ⁺ , formed by the activity of monoamine oxidase on MPTP. Inhibitors of monoamine oxidase block the neurotoxicity of MPTP in both mice and primates. The specificity of the neurotoxic effects of MPP ⁺ for dopaminergic neurons appears to be due to the uptake of MPP ⁺ by the synaptic dopamine transporter. Blockers of this transporter prevent MPP ⁺ neurotoxicity. MPP ⁺ has been shown to be a relatively specific inhibitor of mitochondrial complex I activity, binding to complex I at the retenone binding site and impairing oxidative phosphorylation. In vivo studies have shown that MPTP can deplete striatal ATP concentrations in mice. It has been demonstrated that MPP administered intrastriatally to rats produces significant depletion of ATP as well as increased lactate concentration confined to the striatum at the site of the injections. Compounds that enhance ATP production can protect against MPTP toxicity in mice.

A compound of the disclosure is administered to animals such as mice or rats for three weeks before treatment with MPTP. MPTP is administered at an appropriate dose, dosing interval, and mode of administration for 1 week before sacrifice. Control groups receive either normal saline or MPTP hydrochloride alone. Following sacrifice the two striate are rapidly dissected and placed in chilled 0.1 M perchloric acid. Tissue is subsequently sonicated and aliquots analyzed for protein content using a fluorometer assay. Dopamine, 3,4- dihydroxyphenylacetic acid (DOPAC), and ho mo vanillic acid (HVA) are also quantified.

Concentrations of dopamine and metabolites are expressed as nmol/mg protein.

Compounds of the disclosure that protect against DOPAC depletion induced by MPTP, HVA, and/or dopamine depletion are neuroprotective and therefore can be useful for the treatment of Parkinson's disease.

Haloperidol-Induced Hypolocomotion

The ability of a compound to reverse the behavioral depressant effects of dopamine antagonists, such as haloperidol, in rodents is considered a valid method for screening drugs with potential anti-Parkinsonian effects (Mandhane, et al., Eur. J. Pharmacol 1997, 328, 135-141). Hence, the ability of compounds of Formula (I), (II), (III), or (IV) to block haloperidol-induced deficits in locomotor activity in mice can be used to assess both in vivo and potential antiparkinsonian efficacy.

Mice used in the experiments are housed in a controlled environment and allowed to acclimatize before experimental use. One and one-half (1.5) hours before testing, mice are administered 0.2 mg/kg haloperidol, a dose that reduces baseline locomotor activity by at least 50%. A test compound is administered 5-60 min prior to testing. The animals are then placed individually into clean, clear polycarbonate cages with a flat perforated lid. Horizontal locomotor activity is determined by placing the cages within a frame containing a 3x6 array of photocells interfaced to a computer to tabulate beam interrupts. Mice are left undisturbed to explore for 1 h, and the number of beam interruptions made during this period serves as an indicator of locomotor activity, which is compared with data for control animals for statistically significant differences.

6-Hydroxydopamine Animal Model

The neurochemical deficits seen in Parkinson's disease can be reproduced by local injection of the dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA) into brain regions containing either the cell bodies or axonal fibers of the nigrostriatal neurons. By unilaterally lesioning the nigrostriatal pathway on only one-side of the brain, a behavioral asymmetry in movement inhibition is observed. Although unilaterally-lesioned animals are still mobile and capable of self-maintenance, the remaining dopamine-sensitive neurons on the lesioned side become supersensitive to stimulation. This is demonstrated by the observation that following systemic administration of dopamine agonists, such as apomorphine, animals show a pronounced rotation in a direction contralateral to the side of lesioning. The ability of compounds to induce contralateral rotations in 6-OHDA lesioned rats has been shown to be a sensitive model to predict drug efficacy in the treatment of Parkinson's disease.

Male Sprague-Dawley rats are housed in a controlled environment and allowed to acclimatize before experimental use. Fifteen minutes prior to surgery, animals are given an intraperitoneal injection of the noradrenergic uptake inhibitor desipramine (25 mg/kg) to prevent damage to nondopamine neurons. Animals are then placed in an anesthetic chamber and anesthetized using a mixture of oxygen and isoflurane. Once unconscious, the animals are transferred to a stereotaxic frame, where anesthesia is maintained through a mask. The top of the head is shaved and sterilized using an iodine solution. Once dry, a 2 cm long incision is made along the midline of the scalp and the skin retracted and clipped back to expose the skull. A small hole is then drilled through the skull above the injection site. In order to lesion the nigrostriatal pathway, the injection cannula is slowly lowered to position above the right medial forebrain bundle at -3.2 mm anterior posterior, -1.5 mm medial lateral from the bregma, and to a depth of 7.2 mm below the dura mater. Two minutes after lowering the cannula, 6-OHDA is infused at a rate of 0.5 μΙ ηϊη over 4 min, to provide a final dose of 8 μg. The cannula is left in place for an additional 5 min to facilitate diffusion before being slowly withdrawn. The skin is then sutured shut, the animal removed from the sterereotaxic frame, and returned to its housing. The rats are allowed to recover from surgery for two weeks before behavioral testing. Rotational behavior is measured using a rotameter system having stainless steel bowls (45 cm dia x 15 cm high) enclosed in a transparent Plexiglas cover around the edge of the bowl and extending to a height of 29 cm. To assess rotation, rats are placed in a cloth jacket attached to a spring tether connected to an optical rotameter positioned above the bowl, which assesses movement to the left or right either as partial (45°) or full (360°) rotations.

To reduce stress during administration of a test compound, rats are initially habituated to the apparatus for 15 min on four consecutive days. On the test day, rats are given a test compound, e.g., a compound of Formula (I), (II), (III), or (IV). Immediately prior to testing, animals are given a subcutaneous injection of a sub-threshold dose of apomorphine, and then placed in the harness and the number of rotations recorded for one hour. The total number of full contralateral rotations during the hour test period serves as an index of anti-Parkinsonian drug efficacy.

Description 10

Animal Model for Assessing Therapeutic Efficacy of Monomethyl and monoethyl fumarate

Prodrugs for Treating Alzheimer's Disease

Heterozygous transgenic mice expressing the Swedish AD mutant gene, hAPPK670N, M671L (Tg2576; Hsiao, Learning & Memory 2001 , 8, 301-308) are used as an animal model of Alzheimer's disease. Animals are housed under standard conditions with a 12: 12 light/dark cycle and food and water available ad libitum. Beginning at 9 months of age, mice are divided into three groups. The first two groups of animals receive increasing doses of a compound of Formula (I), (II), (III), or (IV), over six weeks. The remaining control group receives daily saline injections for six weeks.

Behavioral testing is performed at each drug dose using the same sequence over two weeks in all experimental groups: (1) spatial reversal learning, (2) locomotion, (3) fear conditioning, and (4) shock sensitivity.

Acquisition of the spatial learning paradigm and reversal learning are tested during the first five days of test compound administration using a water T-maze as described in Bardgett et ah, Brain Res Bull 2003, 60, 131-142. Mice are habituated to the water T-maze during days 1-3, and task acquisition begins on day 4. On day 4, mice are trained to find the escape platform in one choice arm of the maze until 6 to 8 correct choices are made on consecutive trails. The reversal learning phase is then conducted on day 5. During the reversal learning phase, mice are trained to find the escape platform in the choice arm opposite from the location of the escape platform on day 4. The same performance criteria and inter-trial interval are used as during task acquisition.

Large ambulatory movements are assessed to determine that the results of the spatial reversal learning paradigm are not influenced by the capacity for ambulation. After a rest period of two days, horizontal ambulatory movements, excluding vertical and fine motor movements, are assessed in a chamber equipped with a grid of motion-sensitive detectors on day 8. The number of movements accompanied by simultaneous blocking and unblocking of a detector in the horizontal dimension are measured during a one-hour period.

The capacity of an animal for contextual and cued memory is tested using a fear conditioning paradigm beginning on day 9. Testing takes place in a chamber that contains a piece of absorbent cotton soaked in an odor-emitting solution such as mint extract placed below the grid floor. A 5-min, 3 trial 80 db, 2800 Hz tone-foot shock sequence is administered to train the animals on day 9. On day 10, memory for context is tested by returning each mouse to the chamber without exposure to the tone and foot shock, and recording the presence or absence of freezing behavior every 10 seconds for 8 minutes. Freezing is defined as no movement, such as ambulation, sniffing or stereotypy, other than respiration.

On day 11 , the response of the animal to an alternate context and to the auditory cue is tested. Coconut extract is placed in a cup and the 80 dB tone is presented, but no foot shock is delivered. The presence or absence of freezing in response to the alternate context is then determined during the first 2 minutes of the trial. The tone is then presented continuously for the remaining 8 minutes of the trial, and the presence or absence of freezing in response to the tone is determined.

On day 12, the animals are tested to assess their sensitivity to the conditioning stimulus, i.e., foot shock.

Following the last day of behavioral testing, animals are anesthetized and the brains removed, post-fixed overnight, and sections cut through the hippocampus. The sections are stained to image β-amyloid plaques.

Data is analyzed using appropriate statistical methods. Description 11

Animal Model for Assessing Therapeutic Efficacy of Monomethyl and monoethyl fumarate

Prodrugs for Treating Huntington's Disease

Neuroprotective Effects in a Transgenic Mouse Model of Huntington 's Disease

Transgenic HD mice of the N171-82Q strain and non-transgenic littermates are treated with a compound of Formula (I), a compound of Formula (II), or a vehicle from 10 weeks of age. The mice are placed on a rotating rod ("rotarod"). The length of time at which a mouse falls from the rotarod is recorded as a measure of motor coordination. The total distance traveled by a mouse is also recorded as a measure of overall locomotion. Mice administered compounds of the disclosure that are neuroprotective in the N171-82Q transgenic HD mouse model remain on the rotarod for a longer period of time and travel farther than mice administered vehicle.

Malonate Model of Huntington 's Disease

A series of reversible and irreversible inhibitors of enzymes involved in energy generating pathways has been used to generate animal models for neurodegenerative diseases such as Parkinson's and Huntington's diseases. In particular, inhibitors of succinate

dehydrogenase, an enzyme that impacts cellular energy homeostasis, has been used to generate a model for Huntington's disease.

To evaluate the effect of compounds of Formula (I), (II), (III), or (Γν) in this malonate model for Huntington's disease, a compound of Formula (I), (II), (III), or (IV) is administered at an appropriate dose, dosing interval, and route, to male Sprague-Dawley rats. A compound of Formula (I), (II), (III), or (Γν) is administered for two weeks prior to the administration of malonate and then for an additional week prior to sacrifice. Malonate is dissolved in distilled deionized water and the pH adjusted to 7.4 with 0.1 M HC1. Intrastriatal injections of 1.5 μL of 3 μηιοΐ malonate are made into the left striatum at the level of the Bregma, 2.4 mm lateral to the midline and 4.5 mm ventral to the dura. Animals are sacrificed at 7 days by decapitation and the brains quickly removed and placed in ice cold 0.9% saline solution. Brains are sectioned at 2 mm intervals in a brain mold. Slices are then placed posterior side down in 2% 2,3,5- tiphenyltetrazolium chloride. Slices are stained in the dark at room temperature for 30 min and then removed and placed in 4% paraformaldehyde pH 7.3. Lesions, noted by pale staining, are evaluated on the posterior surface of each section. The measurements are validated by comparison with measurements obtained on adjacent Nissl stain sections. Compounds exhibiting a neuroprotective effect and therefore potentially useful in treating Huntington's disease show a reduction in malonate-induced lesions.

Description 12

Animal Model for Assessing Therapeutic Efficacy of Monomethyl and monoethyl fumarate

Prodrugs for Treating Amyotrophic Lateral Sclerosis

A murine model of SOD1 mutation-associated ALS has been developed in which mice express the human superoxide dismutase (SOD) mutation glycine— ^alanine at residue 93

(SOD1). These SOD1 mice exhibit a dominant gain of the adverse property of SOD, and develop motor neuron degeneration and dysfunction similar to that of human ALS. The SOD1 transgenic mice show signs of posterior limb weakness at about 3 months of age and die at 4 months.

Features common to human ALS include astrocytosis, microgliosis, oxidative stress, increased levels of cyclooxygenase/prostaglandin, and, as the disease progresses, profound motor neuron loss.

Studies are performed on transgenic mice overexpressing human Cu/Zn-SOD G93A mutations (B6SJL-TgN (SOD1-G93A) 1 Gur) and non-transgenic B6/SJL mice and their wild litter mates. Mice are housed on a 12-hr day/light cycle and (beginning at 45 d of age) allowed ad libitum access to either test compound-supplemented chow, or, as a control, regular formula cold press chow processed into identical pellets. Genotyping can be conducted at 21 days of age as described in Gurney et ah, Science 1994, 264(5166), 1772-1775. The SOD1 mice are separated into groups and treated with a test compound, e.g., compound of Formula (I), (II), (III), or (IV), or serve as controls.

The mice are observed daily and weighed weekly. To assess health status mice are weighed weekly and examined for changes in lacrimation/salivation, palpebral closure, ear twitch and pupillary responses, whisker orienting, postural and righting reflexes and overall body condition score. A general pathological examination is conducted at the time of sacrifice.

Motor coordination performance of the animals can be assessed by one or more methods known to those skilled in the art. For example, motor coordination can be assessed using a neurological scoring method. In neurological scoring, the neurological score of each limb is monitored and recorded according to a defined 4-point scale: 0 - normal reflex on the hind limbs (animal will splay its hind limbs when lifted by its tail); 1 - abnormal reflex of hind limbs (lack of splaying of hind limbs when animal is lifted by the tail); 2 - abnormal reflex of limbs and evidence of paralysis; 3 - lack of reflex and complete paralysis; and 4 - inability to right when placed on the side in 30 seconds or found dead. The primary end point is survival with secondary end points of neurological score and body weight. Neurological score observations and body weight are made and recorded five days per week. Data analysis is performed using appropriate statistical methods.

The rotarod test evaluates the ability of an animal to stay on a rotating dowel allowing evaluation of motor coordination and proprioceptive sensitivity. The apparatus is a 3 cm diameter automated rod turning at, for example, 12 rounds per min. The rotarod test measures how long the mouse can maintain itself on the rod without falling. The test can be stopped after an arbitrary limit, for example at 120 sec. If the animal falls down before 120 sec, the performance is recorded and two additional trials are performed. The mean time of 3 trials is calculated. A motor deficit is indicated by a decrease of walking time.

In the grid test, mice are placed on a grid (length: 37 cm, width: 10.5 cm, mesh size: l xl cm ) situated above a plane support. The number of times the mice put their paws through the grid is counted and serves as a measure for motor coordination.

The hanging test evaluates the ability of an animal to hang on a wire. The apparatus is a wire stretched horizontally 40 cm above a table. The animal is attached to the wire by its forepaws. The time needed by the animal to catch the string with its hind paws is recorded (60 sec max) during three consecutive trials.

Electrophysiological measurements (EMG) can also be used to assess motor activity condition. Electromyographic recordings are performed using an electromyography apparatus. During EMG monitoring mice are anesthetized. The measured parameters are the amplitude and the latency of the compound muscle action potential (CMAP). CMAP is measured in

gastrocnemius muscle after stimulation of the sciatic nerve. A reference electrode is inserted near the Achilles tendon and an active needle placed at the base of the tail. A ground needle is inserted on the lower back of the mice. The sciatic nerve is stimulated with a single 0.2 msec pulse at supramaximal intensity (12.9 niA). The amplitude (mV) and the latency of the response (ms) are measured. The amplitude is indicative of the number of active motor units, while distal latency reflects motor nerve conduction velocity.

The efficacy of test compounds can also be evaluated using biomarker analysis. To assess the regulation of protein biomarkers in SOD1 mice during the onset of motor impairment, samples of lumbar spinal cord (protein extracts) are applied to ProteinChip Arrays with varying surface chemical/biochemical properties and analyzed, for example, by surface enhanced laser desorption ionization time of flight mass spectrometry. Then, using integrated protein mass profile analysis methods data is used to compare protein expression profiles of the various treatment groups. Analysis can be performed using appropriate statistical methods.

Description 13

Animal Model for Assessing GI Irritation of Monomethyl and monoethyl fumarate

Prodrugs

At least one MMF prodrug, e.g., dimethyl fumarate, is known to cause gastrointestinal irritation. The Annamalai-Ma gastrointestinal irritation rat model is predictive of gastrointestinal irritation of MMF prodrugs in humans. This animal model has several common features of other published GI irritation animal models including the Whiteley-Dalrymple model described in Models of Inflammation: Measuring Gastrointestinal Ulceration in the Rat, Pharmacology (1998) 10.2.1-10.2.4; as well as the animal models disclosed in Joseph J. Bertone, DVM, MS,

DipACVIM. Prevalence of Gastric Ulcers in Elite, Heavy Use Western Performance Horses, AAEP Proceedings / Vol. 46 / 2000; and isbil Buyukcoskun N., Central Effects of Glucagon-like Peptide- 1 on Cold Restraint Stress-induced Gastric Mucosal Lesions, Physiol. Res. 48: 451-455, 1999.

In order to assess gastrointestinal irritation using this model, rats are treated orally with either vehicle or the MMF prodrug of the present disclosure (n = 10 per group) at 180 mg- equivalents MMF/kg of animal body weight, dosed once per day for 4 days, followed by necropsy and gastrointestinal evaluation at 24 hrs after the final dose. Evans Blue dye is injected IV (tail vein) to visually emphasize any lesions in the gastrointestinal tissue.

Accordingly, rats are dosed once per day for 4 consecutive days with 180 mg-equivalents MMF / kg body weight per day. The animals are fasted overnight prior to necropsy. On Day 5, to help visualize lesions, 1 mL of 1% Evan's blue in saline is injected into the tail vein 30 minutes prior to euthanasia. The animals are euthanized by inhalation of carbon dioxide. A partial necropsy, limited to the abdominal cavities, is then performed. The stomach and small intestine are removed. Residual material is washed away, using an irrigation syringe filled with saline. The stomach is cut along the larger curvature and washed gently with normal saline, and is examined for any lesions. The stomachs are scored in accordance with the scoring system outlined in Table 4.

Table 4: Scoring System for Stomach Lesions in the Rat

Finally, it should be noted that there are alternative ways of implementing the embodiments disclosed herein. Accordingly, the present embodiments are to be considered as illustrative and not restrictive, and the claims are not to be limited to the details given herein, but may be modified within the scope and equivalents thereof.

From the foregoing description, various modifications and changes in the compositions and methods of this disclosure will occur to those skilled in the art. All such modifications coming within the scope of the appended claims are intended to be included therein.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

At least some of the chemical names of compounds of the disclosure as given and set forth in this application, may have been generated on an automated basis by use of a

commercially available chemical naming software program, and have not been independently verified. In the instance where the indicated chemical name and the depicted structure differ, the depicted structure will control.

Chemical structures shown herein were prepared using ChemDraw or ISIS ^® /DRAW. Any open valency appearing on a carbon, oxygen or nitrogen atom in the structures herein indicates the presence of a hydrogen atom. Where a chiral center exists in a structure but no specific stereochemistry is shown for the chiral center, both enantiomers associated with the chiral structure are encompassed by the structure.