Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
  • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
  • C07D307/26—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D307/30—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D307/32—Oxygen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
  • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
  • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
  • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D307/66—Nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase

Definitions

• the present invention relates to compounds which modulate the level or activity of one or more polypeptides having Fe(!l) and 2-oxoglutarate (20G)-dependent oxygenase activity in a cell, tissue, or subject, exemplified by the Fat Mass and Obesity Associated protein (FTO).
• FTO Fat Mass and Obesity Associated protein
• the invention is also directed to compositions comprising compounds which modulate the activity of FTO, including pharmaceuticaiiy-acceptab!e compositions, and to methods of preventing, ameliorating, or treating FTO-mediated conditions or diseases in a subject, including stroke, epilepsy, depression, and degenerative central nervous system disorders such as Alzheimer's and Parkinson's disease.
• Other aspects of the invention relate to use of the compounds to ameliorate or treat conditions associated with metabolic disorders, such as diabetes and obesity, respectively.
• Epilepsy is a chronic disorder of abnormal electrical activity in the brain characterized by recurrent unprovoked seizures (Rogers, S. j.; Cavazos, j. E. (2011) In: Pharmacotherapy, A Pathophysiologic Approach; DiPiro j. T. et al ., Eds., McGraw-Hill: New York, 979-1005). It manifests as a collection of disorders which vary widely in etiology, appearance., and severity. Treatment with traditional antiepileptic drugs I ' AED) may reduce symptoms of seizures but does not influence the course of the disorder. Despite some benefit of AED treatment, approximately 30-35% of treated patients will be refractory (Rogers, S. J.; Cavazos, j.
• Epilepsy is thought to be caused by some predisposing factor(s) (e.g., genetic, age, environmental) and/or insult (e.g., head trauma, status epilepticus, febrile seizure) that trigger the initial seizure.
• predisposing factor(s) e.g., genetic, age, environmental
• insult e.g., head trauma, status epilepticus, febrile seizure
• brain neuronal circuitry undergoes changes that may eventually produce a condition of chronic, spontaneous seizures, i.e., epilepsy. This lag time may be 1-2 weeks In animal models, or up to several years in humans.
• Epi!eptogenesis refers to the time period between the initial insult and spontaneous, recurring seizures. More recently it has been extended to include both this initial latency period up to the point of epilepsy, as well as the progression of events that occur after epilepsy has been established (Pitkanen, A. (2010), Epilepsia, 51(S3):2-17; Pitkanen, A.; Lukasiuk, K. (2011), Lancet Neurol., 10 :173-186). Antleplleptogenic agents therefore would have such activities as decreasing seizure frequency and severity, increasing responsiveness to traditional AEDs, and decreasing pharmacoresistance ( ani, R.; Pollard, J.; Dichter, . A. (2011), Neurosci. Lett., 497:251-256).
• the gene fat mass and obesity-associated (FTO) was first identified in mice as one of the genes encoded by the 1.6 Mb deletion that produced a phenotype with partial syndactyly of fore!imbs and extensive thymic hyperplasia (Peters et al.. a mm Genome 1999; 10:983-6). Subsequently, a common variant in the FTO gene was identified as a risk allele for type 2 diabetes and increased body mass (Frayling et al.. Science 2007; 316:889-94), and many studies of FTO have focused on the association with diabetes, obesity, and metabolism (Larder et a!., Trends Endocrinol Metab 2011; 22:53-9).
• FTO central nervous system
• mice that were specifically deleted only for neuronal FTO had a similar phenotype of growth retardation as the whole body FTO deletion, suggesting that a major function of FTO occurs in the brain (Gao et al, PLoS One 2010; 5:el4005).
• the obesity- associated risk allele has been shown to have a potential pathological effect on brain volume; healthy elderly subjects with the risk allele had brain volume deficits (average differences of 8% in frontal lobes and 12% in occipital lobes) compared to non-carriers (Ho et a!., Proc Natl Acad Sci U S A 2010; 107:8404- 9).
• BDNF brain derived neurotrophic factor
• One aspect of the invention is related to a compound of Formii!a i, a pharmaceuticaliy- acceptable salt thereof, and mixtures of any of the forgoing
• Ar is a substituted or unsubstituted aryl or heteroaryi ring
• said aryl ring comprises at least one R group selected from the group consisting of -H, - OH, -SH, -CN, -F, -CI, -Br, -CN, N0 2 -NO, -NH 3 , -NHR, -NRR, -C(0)R, -C(0)OH, -C(0)OR, -C(0)NH 2 , -C(0)NHR, and -C(0)NRR, -SOR, -S0 2 R, -S0 2 MH 2 , -S0 2 NHR, -S0 2 NRR;
• each R is independently selected from the group consisting of (CVC 5 ) aiky!, substituted (Ci-Q) alky!, (C 2 -C 6 ) alkeny!, substituted (C 2 -Q) aikeny!, (C 2 -C e ) alkyny!, substituted (C 2 -Q) a!kyny!, !C 5 -C 20 ) aryl, substituted (C 3 -C 20 ) aryl, (C 6 -C 26 ) arylalky!, substituted (C 6 -C 26 ) aryialkyi, 5-20 membered heteroaryi, substituted 5-20 membered heteroaryi, 6-26 membered heteroarylaikyl, and substituted 6-26 membered heteroaryialkyl; and
• V is selected from the group consisting of -S0 2 R, - S0 2 R, -C(0)R, -C(0)OR, -C(S)R, and -
• compositions including pharmaceutical compositions, comprising the compounds of the invention, noted above, including method for pharmaceutical formulation of previously described compounds for use in oral and intravenous applications, and in implantable materials.
• One aspect of the invention is directed to a method of preventing, ameliorating, or treating at least one FTO-mediated condition or disease in a subject, comprising administering to said subject a therapeuticai!y-effective amount of the compound or salt described above and encompassed by any of Formulas !-V!!, wherein said condition or disease is selected from the group consisting of neurological disorders, degenerative central nervous system disorders, metabolic disorders, and proliferative cell disorders.
• One aspect of the invention relates to a method of modulating the level or activity of at least one FTO protein in a cell, comprising contacting the cell with an effective amount of the compound or salt described above and encompassed by any of Formulas i-V!i.
• One aspect of the invention relates to a method of modulating the level or activity of at least one FTO protein in a subject, which comprises administering a therapeuticai!y-effective amount of an effective amount of the compound or salt described above and encompassed by any of Formulas !-V!S to said subject.
• One aspect of the invention is directed to the treatment of CMS diseases by modulating mRNA N6-methy!adenosine (m6A), another aspect of the invention is through modulation of microRNA (miRNA), and another aspect of the invention is through the rnoduiation of mRNA expression.
• Panel A sets forth an illustration showing the mechanism of FTO modulation of m6A.
• FTO is known to demethyiate non-stoichiometricai!y, for example resulting in 20.. 50, or 70% demethylation of appropriate substrates.
• Panel B illustrates the consequences of FTO modulation of m6A in mRNA.
• FiG. 2 sets forth the synthesis of FTO modulators.
• the following legends describe components used in each of the named reaction schemes: Synthetic schemes for the synthesis of compounds la-az through ISa-az, Reactions a-g are as follows: (a) KCN, glyoxal, Na2C03, H20; (b) D AP, CIS02R1, DMF, THF; (c) DMAP, CIS02CH2CH2CH2CI, DMF, THF; (d) aH, THF; (e) CH2N2, MeOH; if) TEA, CISiMe2tBu, THF, CICOR2, DMAP, DMF, Bu4NF; (g) TEA, C!SiMe2tBu, THF, CiCOOEt, DMAP, DMF, Bu4MF; (h) SCNR3, THF.
• F!G. 3 sets forth an illustration showing the neuroprotective effects of FTO modulators.
• Mesy! tetronimide represents compound 2c
• Ethyl tetronimide represents compound 3c.
• HCA is homocysteinic acid and is used to simulate oxidative neurological stress.
• F!G. 4 sets forth an illustration showing the inhibition of FTO by modulators 2c (inhibitor 3 ⁇ and 3c (Inhibitor 4).
• FiG. 5 sets forth an illustration showing the modulation of celisjiar m6A by 3c (inhibitor).
• aa or AA amino acid
• RT room temperature
• U units
• TEA triethyiamine
• LDA lithium diisopropyl amine
• DMAP 4-dimethylaminopyridine
• DMF N,N'-dimethyiformamide.
• ceil and “ceils”, which are meant to be inclusive, refer to one or more cells which can be in an isolated or cultured state, as in a ceil line comprising a homogeneous or heterogeneous population of cells, or in a tissue sample, or as part of an organism, such as a transgenic mammal.
• amino acid encompasses both naturally occurring and non-naturally occurring amino acids unless otherwise designated.
• an effective amount means an amount of the substance in question which produces a statistically significant effect.
• an “effective amount” for therapeutic uses is the amount of the composition comprising an active compound herein required to provide a clinically significant alteration in a measurable trait.
• effective amounts will be determined using routine optimization techniques and are dependent on the particular condition to be treated, the condition of the patient, the route of administration, the formulation., and the judgment of the practitioner and other factors evident to those skilled in the art.
• the dosage required for the compounds of the invention is manifested as that which induces a statistically significant difference between treatment and control groups.
• a “therapeutica!y-effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
• a therapeutically-effective amount of modulator may vary according to factors such as the disease state, age, sex, and weight of the individual., and the ability of the modulator to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.
• a therapeutically-effective amount is also one in which any toxic or detrimental effects of the modulator are outweighed by the therapeutically beneficial effects.
• a prophylactlcaiiy effective amount refers to an amount effective, at dosages and for periods of time necessary., to achieve the desired prophylactic result.
• a prophy!actica!ly effective amount can be determined as described above for the therapeutically-effective amount.
• the prophylactica!y effective amount will be less than the therapeutically-effective amount.
• treatment includes both prophylaxis and therapy.
• animal includes human beings.
• substituted aromatic or heteroaromatic refers to aromatic or heteroaromatic rings may contain one or more substituents such as -OH, SH, -CM, -F, -CI, -Br, -R, -M0 - O, -NH 2 , -NHR, -MRR, - C(0)R, -C(0)OH, -C(0)OR, -C(0)NH 2 , -C(0)NHR, -CfOJNRR, and the like where each R is independently (C Cs) alkyl, substituted (C C £ ) aikyl, (C 2 -C 6 ) aikeny!, substituted (C 2 -C 6 ) alkenyl, (C -C 6 ) a!kynyl, substituted (C 2 -C 5 ) a!kynyl, (C 5 -C 20 ) aryl, substituted (C 5 -C 20 ) ary!, (C 5 -C 20
• a “derivative" of a compound X refers to a form of X in which one or more reactive groups on the compound have been derivatized with a substituent group.
• Peptide derivatives include pep tides in which an amino acid side chain, the peptide backbone, or the amino' or carboxy-terminus has been derivatized (e.g., peptidic compounds with 5 methylated amide linkages).
• An "analogue” of a compound X refers to a compound which retains chemical structures of X necessary for functional activity of X yet which also contains certain chemical structures which differ from X.
• An analogue of a naturally-occurring peptide is a peptide which includes one or more non-naturaily- occurring amino acids.
• mimetic refers to a compound having similar functional and/or structural properties to another known compound or a particular fragment of that known compound.
• a “mimetic" of a compound X refers to a compound in which chemical structures of X necessary for functional activity of X have been replaced with other chemical structures which mimic the conformation of X.
• mimetic, and in particular, peptido imetic is intended to include isosteres.
• cyclic group is intended to include cyclic saturated or unsaturated (i.e., aromatic) group having from about 3 to 10, preferably about 4 to 8, and more preferably about 5 to 7, carbon atoms.
• exemplary cyclic groups include cyciopropyi, cyclobutyl, cyclopentyi, cyclohexy!, and cyclooctyl. Cyclic groups may be unsubstituted or substituted at one 35 or more ring positions.
• a cyclic group may be substituted with halogens, alkyls, cyc!oaikyls, alkeny!s, alkynyls, aryls, heterocycles, hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyis, si!y!s, ethers, thioethers, sulfony!s, sulfonates, selenoethers, ketones, aldehydes, esters, 'CF 3 , 'CN, or the like.
• heterocyclic group is intended to include cyclic saturated or unsaturated (i.e., aromatic) group having from about 3 to 10, preferably about 4 to 8, and more preferably about 5 to 7, carbon atoms, wherein the ring structure includes about one to four heteroatoms.
• Heterocyclic groups include pyrrolidine, oxoiane, thiolane, imidazole, oxazo!e, piperidine, piperazine, morpholine and pyridine.
• the heterocyclic ring can be substituted at one or more positions with such substituents as, for example, halogens, alkyls, cyc!oalkyis, a!kenyls, alkynyls, aryis, other heterocycles, hydroxy!, amino, nitro thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyis, eilyls, ethers, thioethers, su!fonyis, selenoethers, ketones, aldehydes, esters, 'CF 3 , 'CM, or the like.
• Heterocycles may also be bridged or fused to other cyclic groups as described below.
• po!ycyclic group as used herein is intended to refer to two or more saturated or unsaturated (i.e., aromatic) cyclic rings in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings.
• Each of the rings of the po!ycyclic group can he substituted with such substituents as described above, as for example, halogens, alkyls, cycloa!kyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyis, silyis, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, 'CF 3 , 'CN, or the like.
• substituents as described above, as for example, halogens, alkyls, cycloa!kyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyis
• modifying group and "modifying group” are used interchangeably to describe a chemical group directly or indirectly attached to a peptidic structure.
• a modifying group(s) can be direciiy attached by covalent coupling to the peptidic structure or a modifying group(s) can be attached indirectly by a stable non-covalent association.
• composition of matter stand equally well for the enzyme modulators or inhibitors described herein, including all enantiomeric forms, diastereomeric forms, salts, and the like.
• the terms “compound,” “analog,” and “composition of matter” are used interchangeably throughout the present specification.
• the compounds disclosed herein include all salt forms, for example, salts of both basic groups, inter alia, amines, as weli as salts of acidic groups, inter alia, carboxyiic acids.
• the following are non- limiting examples of anions that can form pharmaceutically acceptable salts with basic groups: chloride, bromide, iodide, sulfate, bisuifate, carbonate, bicarbonate, phosphate, formate, acetate, propionate, butyrate, pyruvate, lactate, oxalate, malonate, ma!eate, succinate, tartrate, fumarate, citrate, and the like.
• One aspect of the invention is related to a compound of Formula !, a pharmaceuticaliy- acceptable salt thereof, and mixtures of any of the forgoing
• Ar is a substituted or unsubstituted aryl or heteroaryl ring
• said aryl ring comprises at least one R group selected from the group consisting of -H, - OH, -SH, -CM, -F, -CI, -Br, -CN, NO, -NO, -NH 2 , -NHR, -NRR, -C(0) , -C(0)OH, -C(0)OR, -C(0)NH 2 , -C(0 ⁇ NHR, and -C(0)NRR, -SOR, ⁇ S0 2 R, -S0 2 NH 2 , -S0 2 NHR, -S0 2 NRR;
• each R is independently selected from the group consisting of (Ci-C 5 ) aikyl, substituted (d-Cs) aikyl (C 2 -C 6 ) alkenyi, substituted (C 2 -Q) alkeny!, (C,-C 6 ) a!kyny!, substituted (C 2 -C 6 ) aikynyi, (C 5 -C 2 o) aryl, substituted (C 5 -C 20 ) aryl, (CrC 26 ) aryla!ky!, substituted (C 6 -C 26 ) arylalkyl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered heteroary!aikyl, and substituted 6-26 membered heteroarylalkyl; and
• V is selected from the group consisting of -S0 2 R, - S0 2 R, -C(0)R, -C(0)OR, -C ⁇ S)R, and -
• R 20 , R 21 , and R 22 are independently selected from the group consisting of H, -F, -CI, -Br, -I, -OH, -SH, -CN, N0 2 -NO, - ⁇ ,. -NHR, -NRR, -C(0)R, -C(0)OH, -C(0)OR, -C(0)MH2, -C(0)NHR, -C(0)NRR, - SOR, -SOjR, -S0 2 NH 2 , -SO.NHR, and -S0 2 NRR;
• each R is independently selected from the group consisting of (Ci-C 5 ) aikyi, substituted (CrQ) alkyi, (C 2 -C 6 ) alkeny!, substituted (C 2 -C 6 ) aikeny!, (C 2 -C 6 ) a!kyny!, substituted iC 2 -C 5 ) alkynyl, (C 5 -C 20 ) aryi, substituted (C 5 -C 20 ) aryi, (C «-C 26 ) arylalkyi, substituted (C5-C26) arylalkyi, 5-20 membered heteroaryi, substituted 5-2.0 membered heteroaryi, 6-26 membered heteroarylalkyl, and substituted 6-25 membered heteroarylalkyl; and
• U is selected from the group consisting of hydrogen, (Cj-C 5 ) alky!, substituted (C C 6 ) alkyi, (C 2 -C 6 ) alkenyl, substituted (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, substituted (C 2 -C 6 ) alkynyl, (C 5 -C 20 ) aryi, substituted (C 5 -C 20 ) ary!, (Q-C ⁇ ) arylalkyi, substituted (C 6 -C 2 .-,; arylalkyi, 5-20 membered heteroaryi, substituted 5-20 membered heteroaryi, 5-26 membered heteroarylalkyl, and substituted 5-2.6 membered heteroarylalkyl;
• V is selected from the group consisting of -S0 2 R, - S0 2 R, -C(0)R, -C(0)OR, -C ⁇ S)R, and -
• Z 1 , Z 2 , Z 3 , ⁇ t are independently selected from C, N;
• Another aspect of the invention relates to a compound represented by the structure of Formuia
• R 20 , R 21 , and R 22 are independently selected from the group consisting of H, -F, -CI, -Br, -I, -OH, -SH, -CM, N0 2 -NO, - H 2 . -NHR, -NRR, -C(0)R, -C(0)OH, -C(0)OR, -C(0)NH2, -C(0)NHR, ⁇ C(0)NRR, - SOR, -SO,R, -S0 2 NH 2 , -S0 2 NHR, and -S0 2 NRR;
• Z 1 , Z 2 , Z 3 , Z 4 , 1 ' are independently selected from C, N;
• R J ° is selected from the group consisting of hydrogen, (Ci-C 5 ) a!ky!, substituted (C C 5 ) aikyi, (C 2 -Cs) aikenyl, substituted (C 2 -C 6 ) a!kenyl, (C 2 -Q) aikynyi, substituted (QrQ) alkyny!, (CrC 20 ) aryl, substituted (C 5 -C 20 ) aryl, (C 6 -C 26 ) aryiaikyl, substituted ⁇ Q-C 2S ) aryiaikyl, 5-20 membered heteroaryi, substituted 5-20 membered heteroaryi, 6-26 membered heteroaryialkyl or substituted 6-26 membered heteroarylalky! ;
• W is selected from the group consisting of hydrogen, aikyi, haloa!kyl, -C(0)R, -C(0)OR, - SOR, -S0 2 R, -C(0)NHR, -C(0)NRR where each R is independently (C C 5 ) aikyi, substituted (C C 6 ) alky!, (C 2 - C s ) aikenyl, substituted (C 2 -C 6 ) aikenyl, (C 2 -C 6 ) alkynyi, substituted (C 2 -C 6 ) aikynyi, (C 5 -C 20 5 aryl, substituted (C 5 -C 20 ) aryl, (Q-C 26 ) aryiaikyl, substituted (C6-C26) aryiaikyl, 5-20 membered heteroaryi, substituted 5-20 membered heteroaryi, 5-26 membered heteroaryialkyl or substituted
• Another aspect of the invention relates to a compound represented by the structure of Formula iV:
• R 20 , R 2i , and R 22 are independently selected from the group consisting of H, -F, -CI, -Br, -I, -OH, -SH, -CM, N0 2 -NO, -NH 2 . -NHR, -NRR, -C(0)R, -C(0)OH, -C(0)OR, -C(0)NH2, -C(0)NHR, -CIOjNRR, - SOR, -S0 2 R, -S0 2 NH 2 , -S0 2 NHR, and -S0 2 NRR;
• X is selected from the group consisting of S0 2 R,
• each R is independently (C C 5 ) alkyl, substituted (C-,-C 6 ) a!kyl, (C 2 -C 6 ) aikenyl, substituted (C,-C 5 ) aikenyl, (C,-C 5 ) aikynyi, substituted (C 2 -C 6 ) aikynyi, (C 5 -C 2a ) aryl, substituted (C 5 -C 20 ) aryl, (C 6 -C 26 ) aryiaikyl, substituted (C 6 -C 26 ) aryiaikyl, 5-20 membered heteroaryi, substituted 5-20 membered heteroaryi, 6-26 membered heteroaryialkyl or substituted 6-26 membered heteroaryialkyl.
• Another aspect of the invention relates to a compound represented by the structure of Formula
• R 20 , R 2i , and R 22 are independently selected from the group consisting of H, -F, -CI, -Br, -I, -OH, -SH, -CN, N0 2 -NO, -NH 2 . -N H R, -N RR, -C(0)R, -C(0)OH, -C(0)OR, -C(0)N H 2 , -C(0)rxi H R, -C(0)N RR, - SOR, -S0 2 R, -50 2 N H 2 , -S0 2 NH R, and -50 2 !M RR; and
• I 1 , Z 2 , Z 3 , Z 4 are independently selected from C, N;
• Y is selected from the group consisting of S0 2 R,
• each R is independently (C C 5 ) alkyl, substituted (C-,-C 6 ) a!kyl, (C 2 -C 6 ) alkenyi, substituted (C 2 -C 6 ) a!kenyl, (C 2 -C 6 ) alkynyl, and substituted (C 2 -C 6 ) aikyny!.
• Ar 1 Is selected from the grou p consisting of phenyl, 2-fiuorophenyl, 3-fluorophenyi, 4- fluorophenyi, 2-chlorophenyl, 3-ch!orophenyi, 4-chlorophenyl, 2-hydroxyphenyi, 3-hydroxyphenyl, 4- hydroxylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyi, 2-thiophene, 2-furan, 2-thiazole, l-naphthy!, 2-napthyi, 2-pyridyi, 3-pyridyl, 4-pyridyl, 2-quinolinyl, 3-quinolinyl, 4-quinoiinyl, 2- trlfiuoromethylphenyi, 3-trlfiuoromethylphenyi, 4-trlf!uoromethyiphenyi, 3-cyanophenyl, and 4- cyanophenyl; and
• Y is selected from the group consisting of S0 2 R, where R is independently (Ci-C 5 ) a!kyi, substituted (C C 6 ) alkyl, (C 2 -C 6 ) aikeny!, substituted (C 2 -C 5 ) alkenyi, (C 2 -C 5 ) alkynyl, and substituted (C 2 -C 6 ) alkynyl. by the structure of Formula
• Ar 1 is selected from the group consisting of phenyl, 2-fiuorophenyl, 3-f!uorophenyi, 4- fluorophenyi, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 4-trifluoromethylphenyl, 3-cyanophenyl; and
• Q is selected from the group consisting of S0 2 R, where R is independently (C-,-C 5 ) aikyl, substituted (C C 6 ) alkyl.
• Specific aspects of the invention relate to a compound of Formulas l-V!l selected from the group consisting of the following named compounds: N-(3,4-dihydrQxy-5-(4-chlQrophenyl)-2- furanyl)methanesuifonamide; N-(3,4-dihydroxy-5-(2-ch!orophenyi)-2-furanyl)ethanesuifonamide; N-13,4- dihydrQxy-5-(3-chlQrophenyl)-2-furanyl)ethanesulfonamide; N-(3,4-dihydroxy-5-(4-chiorophenyl)-2- furanyljethanesulfonamide; N-(3,4-dihydroxy-5-(2-fiuorophenyl)-2-furanyl)ethanesulfonamide; N-(3,4- dihydroxy-5-(3-fiuorophenyl)-2-furanyl)ethanesu!fonamide
• Another aspect of the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising at least one pharmaceutically-acceptabie excipient and a therapeutically effective amount of the compound or salt according to any one of Formulas l-V!l, as noted above.
• One aspect of the invention relates to a method of modulating the level or activity of at ieast one polypeptide having Fe(ll) and 2-oxoglutarate (20G) dependent oxygenase activity in a ceil or tissue comprising (a) contacting the ceil with the compound of any of Formulas l-VI!; noted above, and (b) measuring the level or activity of said polypeptide.
• Related aspects include a method of modulating the activity of at Ieast one of said polypeptides in a ceil or tissue wherein said polypeptide is an Fe(li) and 2-oxog!utarate (20G)-dependent oxygenase (Iron 20G dioxygenase domain polypeptide) selected from the group consisting of (a) polypeptide hydroxylases; (b) polypeptide demethylases; and (c) nucleic acid hydroxylases.
• Exemplary polypeptide hydroxylases include F!H, ASPH, PHD, C-P4H, C-P3H, and PLODs (Loenarz and Schofield, 2011).
• Exemplary polypeptide demethylases include lysine demethylases (KDMs) and PHF8.
• Exemplary nucleic acid hydroxylases include FTO and TET1-3.
• Other exemplary demethylases include AlkBHl-8, such as AlkBHS.
• a specific aspect of the invention relates to a method wherein at Ieast one of said polypeptides is FTO.
• the methods of the invention may be carried out using cells or tissues from a variety of sources. Typically these include cells derived from vertebrates, including mammals, particularly primates, and human-derived ceils or tissues.
• One aspect of the invention relates to a method of modulating the level or activity of at least one polypeptide having Fe(!l) and 2-oxoglutarate (20G)-dependent oxygenase activity in a subject, which comprises: (a) administering a therapeutica!!y-effective amount of a compound any of Formulas l-Vil; noted above, to said subject; (b) measuring the level or activity of said polypeptide.
• Related aspects include a method of modulating the activity of at Ieast one of said polypeptides in a subject wherein said polypeptide is an Fe(il) and 2-oxoglutarate ⁇ 20G)-dependent oxygenase (Iron 20G dioxygenase domain polypeptide) selected from the group consisting of (a) polypeptide hydroxylases; (b) polypeptide demethylases; and (c) nucleic acid hydroxylases.
• Exemplary polypeptide hydroxylases include FIH, ASPH, PHD, C-P4H, C-P3H, and PLODs.
• Exemplary polypeptide demethylases include lysine demethylase (KD ) and PHF8.
• Exemplary nucleic acid hydroxylases include FTO and TET1-3.
• a specific aspect of the invention relates to a method wherein at Ieast one of said polypeptides is FTO,
• the subject may be an animal, such as a vertebrate, Including mammals, particularly primates, and humans.
• One aspect of the invention relates to a method of preventing, ameliorating, or treating at least one Fe(il) and 2-oxoglutarate (20G)-dependent oxygenase-mediated condition or disease in a subject, comprising administering to said subject a therapeutically-effective amount of the compound or salt represented by a compound encompassed by any of formulas l-VII.
• Another aspect of the invention relates to a method of preventing, ameliorating, or treating at Ieast one FTO-mediated-mediated condition or disease in a subject, comprising administering to said subject a therapeutically-effective amount of the compound or salt represented by a compound encompassed by any of formulas i-VII, wherein said condition or disease is selected from the group consisting of neurological disorders, degenerative centra! nervous system disorders, metabolic disorders, and drug abuse.
• aspects include methods wherein said condition or disease is a neurological disorder.
• exemplary conditions include but are not limited to epilepsy, epiieptogenesis, depression, and stroke.
• Other aspects relate to methods wherein said condition or disease is a degenerative disorder selected from the group consisting of Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, traumatic brain injury, and multiple sclerosis.
• Still other aspects relate to methods wherein said condition or disease is a metabolic disorder, such as diabetes, anemia, renal failure-associated anemia, and obesity, plus methods wherein said conditions or disease are related to drug substance abuse, drug addiction, and symptoms of drug withdrawal.
• Methods of the invention that relate to administration of a compound or composition comprising a compound represented by any of Formulas !-Vli to a subject may be carried out in a variety of animals, particularly vertebrates, including mammals, particularly primates, and humans. Exemplary mammals also include rodents, such as laboratory mice and rats, plus farm animals, including cows, pigs, goats, sheep, and horses, plus other types of domesticated animals, such as dogs and cats.
• aspects of the invention are directed to the treatment of C S diseases by modulating mRNA N6-methyladenosine (m6A), the modulation of microRNA (miRNA), and the modulation of mRNA expression.
• m6A mRNA N6-methyladenosine
• miRNA microRNA
• Epiieptogenesis is a major cause of epilepsy.
• One major contributor to epilepsy is neuronal death due to an unprovoked seizure, traumatic brain injury, infantile febrile seizures, stroke, or brain insult, Epiieptogenesis may occur after any of these events, leading to an asymptomatic latent period during which the disease state progresses.
• Preventing neuronal death by modulating antioxidant enzyme expression can be accomplished by inhibiting FTO, resulting In a neuroprotective effect.
• One aspect of the invention is directed to a method of preventing, ameliorating, or treating at least one Fe(ll) and 2-oxoglutarate (20G)-dependent oxygenase-mediated condition or disease in a subject, comprising administering to said subject a therapeutica!ly-effective amount of the compound or salt described above and encompassed by any of Formulas l-Vil, wherein said condition or disease is selected from the group consisting of neurological disorders, degenerative central nervous system disorders, metabolic disorders, and proliferative ceil disorders.
• Another aspect of the invention is directed to the methods noted above, wherein said condition or disease is a degenerative disorder selected from the group consisting of Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, traumatic brain injury, and multiple sclerosis.
• condition or disease is a neurological disorder.
• said neurological disorder is selected from the group consisting of epilepsy, epiieptogenesis, depression, and stroke.
• condition or disease is a metabolic disorder, such as obesity, diabetes, anemia, and renal failure-associated anemia.
• condition or disease is a proliferative disease, such as cancer, and specific types of cancer, such as colon cancer.
• Another aspect of the invention is directed to the treatment of C S diseases by modulating neuronal levels of m6A, and microRNA.
• the invention is directed to a method of treating, suppressing, reducing the severity, reducing the risk of developing or inhibiting epilepsy comprising administering a compound of this invention to a subject suffering from epilepsy under conditions effective to treat the epilepsy.
• this invention provides methods for a) treating, suppressing, reducing the severity, reducing the risk, preventing, or Inhibiting epilepsy; b) treating, suppressing, reducing the severity, reducing the risk, preventing, or inhibiting epi!eptogenesis; c) treating, suppressing, reducing the severity, reducing the risk, preventing, or inhibiting temporal lobe epilepsy; d) treating, suppressing, reducing the severity., reducing the risk, preventing, or inhibiting partial seizures; e) treating, suppressing, reducing the severity, reducing the risk, preventing, or inhibiting generalized seizures; f) treating, suppressing, reducing the severity, reducing the risk, preventing, or inhibiting unclassified seizures; g) treating, suppressing, reducing the severity, reducing the risk, preventing, or inhibiting status epiiepticus; h) treating, suppressing, reducing the severity, reducing the risk, preventing, or inhibiting pharmacoresistant seizures
• the compounds of the present invention are useful in the treatment, reducing the severity, reducing the risk, or inhibition of epileptogenesis and drug resistant epilepsy and seizures. Treatment of these different epilepsies is supported by the Examples herein. Moreover, based upon the designed mode of action as Fe(li) and 2-oxoglutarate (20G)-dependent oxygenase inhibitors, it is believed that other CMS disease states, such as depression, schizophrenia, stroke, traumatic brain injury, Alzheimer's and Parkinson's disease will likewise be treatable or preventable upon administration of the compounds or compositions of the present invention to a patient. Preferred compounds of the present invention are able to preserve neurons at risk of death thereby preventing subsequent CNS pathological states due to the death of neurons, and also disrupt the process of epileptogenesis.
• this invention provides for the use of a compound as herein described, or its isomer, metabolite, tautomer, pharmaceutically acceptable salt, pharmaceutical product, polymorph, crystal, M-oxide, hydrate, or any combination thereof, for treating, suppressing, preventing, reducing the severity, reducing the risk, or inhibiting epilepsy in a subject.
• this invention provides for the post-operative recovery and suppression of epileptogenesis following temporal lobe epilepsy surgery.
• temporal lobe epilepsy surgery fails in about 20-30% of patients, with a major cause being the persistence of epileptogenic mesial structures.
• Treatment with a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable carrier under conditions to treat epilepsy is useful by one skilled in the art.
• compositions including pharmaceutical compositions, comprising the compounds of the invention, noted above.
• One aspect of the invention is directed to a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and a therapeutically effective amount of the compound or salt disclosed above.
• Still another aspect of the invention relates to a method for pharmaceutical formulation of previously described compounds for use in oral and intravenous applications, and in implantable materials.
• Another aspect of the present invention relates to a pharmaceutical composition including a pharmaceutically acceptable carrier and a compound according to the aspects of the present invention.
• the pharmaceutical composition can contain one or more of the above-identified compounds of the present invention.
• the pharmaceutical composition of the present invention will include a compound of the present invention or its pharmaceutically acceptable salt, as well as a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier refers to any suitable adjuvants, carriers, excipients, or stabilizers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, emulsions, or implantable disc.
• the composition will contain from about G.01 to 99 percent, preferably from about 20 to 75 percent of active compound(s), together with the adjuvants, carriers and/or excipients. While individual needs may vary, determination of optima! ranges of effective amounts of each component is within the skill of the art.
• Typical dosages comprise about 0.01 to about 100 mg/kg body vvt.
• the preferred dosages comprise about 0.1 to about 100 mg/kg body wt.
• the most preferred dosages comprise about 1 to about 100 mg/kg body wt.
• Treatment regimen for the administration of the compounds of the present invention can aiso be determined readily by those with ordinary skill in art. That is, the frequency of administration and size of the dose can be established by routine optimization, preferably while minimizing any side effects.
• the solid unit dosage forms can be of the conventional type.
• the solid form can be a capsule and the like, such as an ordinary gelatin type containing the compounds of the present invention and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch.
• these compounds are tabulated with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginlc acid, and a lubricant, like stearic acid or magnesium stearate.
• the tablets, capsules, and the like can also contain a binder such as gum tragacanth, acacia, com starch, or gelatin; excipients such as dica!cium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin.
• a liquid carrier such as a fatty oil.
• tablets can be coated with shellac, sugar, or both.
• a syrup can contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
• these active compounds can be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like.
• Such compositions and preparations should contain at least 0.1 % of active compound.
• the percentage of the compound in these compositions can, of course, be varied and can conveniently be between about 2% to about 60% of the weight of the unit.
• the amount of active compound In such therapeutically useful compositions is such that a suitable dosage will be obtained.
• Preferred compositions according to the present invention are prepared so that an oral dosage unit contains between about 1 mg and 800 mg of active compound.
• the active compounds of the present invention may be orally administered, for example, with an inert diluent, or with an assailable edible carrier, or they can be enclosed in hard or soft shell capsules, or they can be compressed into tablets, or they can be incorporated directly with the food of the diet.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, in all cases, the form should be sterile and should be fluid to the extent that easy syrlngability exists, !t should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyoi (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
• the compounds or pharmaceutical compositions of the present invention may also be administered in Injectable dosages by solution or suspension of these materials In a physiologically acceptable diluent with a pharmaceutical adjuvant, carrier or excipient.
• a pharmaceutical adjuvant, carrier or excipient include, but are not limited to, sterile liquids, such as water and oiis, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable components.
• Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
• water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.
• the pharmaceutical forms suitable for implantable use include sterile wafers of polycarboxyphenoxypropane-sebacic-acid (pCPP:SA) polymers, po!y(D,L-lactlc acid), polyhydroxybutyrate, lysine diisocyanate (LDI)-glycerol polyurethane, and poly(D-L lactide-co-giyco!ide).
• pCPP:SA polycarboxyphenoxypropane-sebacic-acid
• LKI lysine diisocyanate
• the form should be sterile and should be a wafer or disc of suitable dimensions for surgical implantation in the brain.
• the polymers should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• the wafers should be biodegradable in the central nervous system, and should permit the slow release of the above mentioned compounds, ranging from 24 hours up to 5 months. Such wafers may be of particular value in enhancing the success of temporal lobe epilepsy surgery by suppressing persistent epileptogenic structures.
• the invention provides compounds and compositions, including any aspect described herein, for use in any of the methods of this invention.
• use of a compound of this invention or a composition comprising the same will have utility in inhibiting, suppressing, enhancing or stimulating a desired response in a subject, as will be understood by one skilled in the art.
• the compositions may further comprise additional active ingredients, whose activity is useful for the particular application for which the compound of this invention is being administered.
• compositions typically must be sterile and stable under the conditions of manufacture and storage.
• the composition can be formulated as a solution, microemu!sion, liposome, or other ordered structure suitable to high drug concentration.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanoi, po!yoi (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• isotonic agents for example, sugars, polyaicohols such as mannitoi, sorbitol, or sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about b including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
• the modulators ca be administered in a time release formulation, for example in a composition 5 which includes a slow release polymer.
• the active compounds can be prepared 'with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, po!yglycollc acid, collagen, polyorthoesters. polyiactic acid and po!yiactic, po!yg!ycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.
• the mode of administration may be oral, for Intestinal delivery; Intranasal, for nasal delivery; and intravenous for delivery through the blood-brain barrier.
• Other modes of administration as are known in the art may also be used, including, but not limited to intrathecal, Intramuscular, intrabronchial, intrarectal, intraocular, and intravagina! delivery,
• the modulator compounds can be administered as oral dosage compositions for small intestinal delivery.
• Such oral dosage compositions for small intestinal delivery are well-known in the art, and generally comprise gastroresistent tablets or capsules (Remington's Pharmaceutical Sciences, 16th Ed., Eds. Osol, Mack Publishing Co., Chapter 89 (1980); Digenis et ai, J. Pharm. Sci., 83:915-921 (1994); Vantini et al, C!inica Terapeutica, 145:445-451 (1393); Yoshitomi et al, Chem. Pharm.
• Tablets are made gastroresistent by the addition of compounds such as cellulose acetate phtha!ate or cellulose acetate terephthalate.
• Capsules are solid dosage forms in which the modulator compound is enclosed in either a hard or soft, soluble container or shell of gelatin.
• the gelatin used in the manufacture of capsules is obtained from collagenous material by hydrolysis. There are two types of gelatin. Type A, derived from pork skins by acid processing, and Type B, obtained from bones and animal skins by alkaline processing.
• Type A derived from pork skins by acid processing
• Type B obtained from bones and animal skins by alkaline processing.
• the use of hard gelatin capsules permit a choice in prescribing a modulator compound or a combination thereof at the exact dosage level considered best for the individual subject.
• the hard gelatin capsule consists of two sections, one slipping over the other, thus completely surrounding the modulator compound.
• capsules are filled by introducing the modulator compound, or gastroresistent beads containing the modulator compound, Into the longer end of the capsule, and then slipping on the cap.
• Hard gelatin capsules are made largely from gelatin, FD&C colorants, and sometimes an opacifying agent, such as titanium dioxide.
• the USP permits the gelatin for this purpose to contain 0.15% (w/v) sulfur dioxide to prevent decomposition during manufacture.
• oral dosage compositions for small intestinal delivery also include liquid compositions which contain aqueous buffering agents that prevent the modulator compound from being significantly inactivated by gastric fluids in the stomach, thereby allowing the modulator compound to reach the small intestines in an active form.
• aqueous buffering agents which can be employed in the present invention include bicarbonate buffer (pH 5.5 to 8.7, preferably about pH 7.4).
• the oral dosage composition is a liquid composition, it is preferable that the composition be prepared just prior to administration so as to minimize stability problems, in this case, the liquid composition can be prepared by dissolving lyophi!ized modulator compound in the aqueous buffering agent.
• Oral dosage compositions for small intestinal delivery also include liquid compositions which may optionally contain aqueous buffering agents that prevent the therapeutic agent and modulator compound from being significantly Inactivated by gastric fluids in the stomach, thereby allowing the biologically active ingredient and modulator compound to reach the small intestines in an active form.
• aqueous buffering agents which can be employed in the present invention include bicarbonate buffer (pH 5.5 to 8.7, preferably about pH 7.4).
• the oral dosage composition is a liquid composition
• the composition be prepared just prior to administration so as to minimize stability problems.
• the liquid composition can be prepared by dissolving iyophilized therapeutic agent and modulator compound in the aqueous buffering agent.
• Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
• a “nasal" delivery composition differs from an “intestinal” delivery composition in that the latter must have gastroresistent properties in order to prevent the acidic degradation of the active agents in the stomach, whereas the former generally comprises water-soluble polymers with a diameter of about 50 ⁇ - ⁇ order to reduce the mucociliary clearance., and to achieve a reproducible bioavailability of the nasally administered agents.
• An “intravenous” delivery composition differs from both the “nasal” and “intestinal” delivery compositions in that there is no need for gastroresistance or water-soluble polymers in the "intravenous" delivery composition.
• Nasal dosage compositions for nasal delivery are well-known in the art.
• Such nasal dosage compositions generally comprise water-soluble polymers that have been used extensively to prepare pharmaceutical dosage forms (Martin et al. In: Physical Chemical Principles of 20 Pharmaceutical Sciences, 3rd Ed., pages 592-638 (1983)) that can serve as carriers for peptides for nasal administration (Davis, In: Delivery Systems for Peptide Drugs, 125:1-21 (1986) ⁇ .
• the nasal absorption of pap tides embedded in polymer matrices has been shown to be enhanced through retardation of nasal mucociliary clearance (Ilium et al, Int. j. Pharm., 46:261-265 (1988).
• the particular water-soluble polymer employed is not critical to the present invention, and can be selected from any of the well-known water-soluble polymers employed for nasal dosage forms.
• a typical example of a water-soluble polymer useful for nasal delivery is polyvinyl alcohol (pvA).
• pvA polyvinyl alcohol
• This material is a swe!lable hydrophilic polymer 'whose physical properties depend on the molecular weight, degree of hydrolysis, cross-linking density, and crystallinity (Peppas et al, In: Hydrogels in Medicine and Pharmacy, 3:109 31 (1987), PYA can be used in the coating of dispersed materials through phase separation, spray-drying, spray-embedding, and spray-densation (Ting et a I, supra).
• a “skin" delivery composition comprising a modulator compound of the invention may include in addition a therapeutic or immunogenic agent, fragrance, creams, ointments, colorings, and other compounds so long as the added component does not de!eterious!y affect transdermal delivery of the therapeutic or immunogenic agent.
• Conventional pharmaceutically acceptable emulsifiers, surfactants, suspending agents, antioxidants, osmotic enhancers, extenders, diluents and preservatives may also be added.
• Water soluble polymers can also be used as carriers.
• the particular therapeutic or immunogenic agent employed is not critical to the present invention, and can be, e.g., any drug compound, biologically active peptide, vaccine, or any other moiety otherwise not absorbed through the transce!!ular pathway, regardless of size or charge.
• the amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form as used herein refers to physically 35 discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
• pharmaceutically-acceptabie carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
• the carrier is 10 suitable for parenteral administration.
• a carrier may be suitable for administration into the central nervous system (e.g., intraspina!y or intracerebrally).
• the carrier can be suitable for intravenous, intraperitoneal or intramuscular administration.
• the carrier is suitable for oral administration.
• Pharmaceutically-acceptabie carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• Mass spectrometry was performed on a Thermo Scientific LTQ-FT at the University of Cincinnati Mass Spectrometry facility.
• the purity of the compounds was monitored by HPLC using a Waters 2695 separation module and a 2487 dual ⁇ absorbance detector with a NovaPak C18 4 ⁇ 3.9x150mm column.
• the mobile phases consisted of acetonitrile/HjO using a 30 minute gradient. All compounds were >95%.
• Microanalysis was performed by Atlantic Microlab Inc., and a!! compounds were found to be ⁇ 0.4%. All reagents were from Sigma-Aldrich.
• Figures 1-5 illustrate the synthetic reactions used to summarize these reactions.
• Table 1 is a non- limiting list of aryl compounds that can be incorporated as "A” from Formula L
• Table 2 illustrates the structures, names, and numbers of a variety of key compounds disclosed in in this application.
• Potassium cyanide (0.91g) was added to sodium carbonate (1.7g) in deionized water (30 mL) in a 3-Neck Glass Round Fiask and placed in an ice bath. The system was repeatedly purged using a vacuum pump and nitrogen gas. Glyoxal (3.72g) was then added to the system without the introduction of 0 2 and the reactants were allowed to dissolve with stirring. In a stoppered tube, the appropriate aryia!dehyde (7.11 mmoles) was added to 1,4-dioxane (5 mL), purged, and then added drop-wise to the system. The system was then removed from the ice bath and allowed to stir at room temperature for 1 hour.
• acetic acid (5 mL) was added drop-wise until gas bubbles were no longer visible from the addition of acetic acid, or until the solution was at a pH of less than 6.
• the soiution was vacuum filtered and washed with ice cold water (5 mL), methanol (5 mL) and ether (5 mL) and then was allowed to air dry. Crude material was recrystal!ized with methanol, collected by vacuum filtration and rinsed with diethyl ether and dried under vacuum.
• N- ⁇ 3,4-dihydroxy-5- ⁇ 2-fluorophenyl)-2-furanyl)ethafiesulfonamide 5g of 5-amino-4-hydroxy-2-(2-f!uorophenyl)-furan-3-one is stirring in 50 mL of dry THF under nitrogen gas for 16 hours with leq dimethyiaminopyridine, 0,5 ml. of DMF and 1 eq of ethanesulfony! chloride. The reaction is filtered, and the filtrate is acidified with saturated ammonium chloride solution, and is extracted 5 times with 20 mL of diethyl ether. The combined ether extracts are washed with brine., and dried with Na 2 S0 4 . After filtering, the solution is concentrated under reduced pressure and recrystaliized with methanol or ethyl acetate.
• 5g of 5-ammo-4-hydroxy-2-(3-f!uorophenyl)-furan-3-orie is stirring in 50 mL of dry THF under nitrogen gas for 16 hours with leq dimethyiaminopyridine, 0.5 mL of DMF and 1 eq of ethanesulfony! chloride.
• the reaction is filtered., and the filtrate is acidified with saturated ammonium chloride solution, and is extracted 5 times with 20 mL of diethyl ether.
• the combined ether extracts are washed with brine, and dried with Na 2 S0 . After filtering, the solution is concentrated under reduced pressure and recrystaliized with methanol or ethyl acetate.
• 5g of 5-amino-4-hydroxy-2-(4-f!uorophenyl)-furan-3-one is stirring in 50 mL of dry THF under nitrogen gas for 16 hours with leq dimethyiaminopyridine, 0.5 mL of DMF and 1 eq of ethanesulfony! chloride.
• the reaction is filtered, and the filtrate is acidified with saturated ammonium chloride solution, and is extracted 5 times with 20 mi. of diethyl ether.
• the combined ether extracts are washed with brine, and dried with NazSO ⁇ . After filtering, the solution is concentrated under reduced pressure and recrysta!!ized with methanol or ethyl acetate.
• 5g of 5-amino-4-hydroxy-2-(4-chiorophenyl)-furan-3-one is stirring In 50 mL of dry THF under nitrogen gas for IS hours with leq dimethyiaminopyridine.. 0.5 mL of DM F and 1 eq of benzenesuifony! chloride. The reaction is filtered, and the filtrate is acidified with saturated ammonium chloride solution, and is extracted 5 times with 20 mi. of diethyl ether. The combined ether extracts are washed with brine, and dried with NazSO ⁇ . After filtering, the solution is concentrated under reduced pressure and recrysta!!ized with methanol or ethyl acetate.
• 5g of 5-amino-4-hydroxy-2-(2-chiorophenyl)-furan-3-orie is stirring in 50 mL of dry THF under nitrogen gas for 16 hours with leq dimethylaminopyridine, 0.5 mL of DMF and 1 eq of i-propanesulfonyi chloride.
• the reaction is filtered, and the filtrate is acidified with saturated ammonium chloride solution, and is extracted 5 times with 20 mL of diethyl ether.
• the combined ether extracts are washed with brine, and dried with Na 2 S0 . After filtering, the solution is concentrated under reduced pressure and recrystailized with methanol or ethyl acetate.
• 5g of 5-amino-4-hydroxy-2-(2-chiorophenyl)-furan-3-one is stirring In 50 mL of dry THF under nitrogen gas for 16 hours with leq dimethylaminopyridine, 0.5 mL of DMF and 1 eq of n-butanesulfonyl chloride.
• the reaction is filtered, and the filtrate is acidified with saturated ammonium chloride solution, and is extracted 5 times with 20 mL of diethyl ether.
• the combined ether extracts are washed with brine, and dried with After filtering, the solution is concentrated under reduced pressure and recrystailized with methanol or ethyl acetate.
• N- ⁇ 3,4-dihydraxy-5- ⁇ 4-chloropheriyl)-2-furariyl)acetamtde 5 g of 5-amino-4-hydroxy-2- ⁇ 4-fluoropheny!2-methyl-furan-3-one is stirring in 50mL dry THF under dry nitrogen.
• the reaction is cooling in an ice bath, and 1 equivalent of triethylamine is added dropwise in dry THF.
• the reaction is warmed to room temperature, chiorotertbutyldimethyisiiane is added dropwise and the reaction is refiuxing gently with a water bath for 30 minutes.
• the reaction is cooled down with an ice bath, and 1 equivalent of acetyl chloride is added dropwise in dry THF.
• reaction is cooling in an ice bath and 1 equivalent of tetrabuty!ammonium fluoride is added and stirs for 30 minutes.
• a saturated ammonium chloride solution is added to quench the reaction, and the reaction is extracted 3 times with 20mL diethyl ether.
• the combined ether extracts are washed with water and brine, the ether is dried with sodium sulfate, is filtered, and evaporates to yield a solid which is recrystallized with methanol.
• Example 1 the biological activity and toxicity of selected compounds disclosed in Example 1 are measured by various assays described below. Mauimai Electroshock (MES) Mode! Assays
• an electrical stimulus of 0.2 s in duration (50 mA in mice and 150 mA in rat at SQHz! is delivered via corneal electrodes primed with an electrolyte solution containing an anesthetic agent. Mice are tested at 30 minutes and 4 hours following doses of 100 mg/kg of test compound. Abolition of the hindiimb tonic extensor component indicates the test compound's ability to inhibit ES- induced seizure spread (White et a!., 1995a; White et a!., 1995b; Swinyard et ai., 1989).
• the scPTZ test utilizes a dose of pentylenetetrazol (85 mg/kg in Carwort Farms Mo. 1 mice). This produces clonic seizures lasting for a period of at least five seconds in 97 per cent (CD97) of animals tested. At the anticipated time of testing the convulsant is administered subcutaneous!y. The test compound is administered intraperitoneal!'/ in mice. Animals are observed over a 30 minute period. Absence of clonic spasms in the observed time period indicates a compound's ability to abolish the effect of pentylenetetrazol on seizure threshold (Swinyard et ai., 1989).
• Test compounds are pre-adrrtinistered to mice via i.p. injection. At varying times, individual mice (four per time point) are challenged with sufficient current delivered through corneal electrodes to elicit a psychomotor seizure in 97% of animals (32 mA for 3s) (Toman et a!., 1952). Untreated mice will display seizures characterized by a minimal clonic phase followed by stereotyped, automatistic behaviors described originally as being similar to the aura of human patients with partial seizures. Animals not displaying this behavior are considered protected. Toxicity is observed using the rotorod test. Sife Mode! Quantitative Assays
• Test compounds are pre-administered to rats via i.p. injection.
• individual rats are challenged with sufficient current delivered through cornea! electrodes to elicit a psychomotor seizure in 97% of animals (32 mA for 3s) (Toman et a!., 1952).
• Untreated mice will display seizures characterized by a minimal clonic phase followed by stereotyped, automatistic behaviors described originally as being similar to the aura of human patients with partial seizures. Animals not displaying this behavior are considered protected. Toxicity Is observed using the rotorod test. Neuroprotection Assay
• the activity assay was performed as previously described ⁇ Fu, Dai et al. 2010, Jia, Fu et al. 2011).
• the reaction mixture contained the following components:
• DNA containing m 6 A (5' ⁇ ATTGTCA(m 6 A)CAGCAGC-3') (SEQ ID NO: 1)
• DMA ' was digested by nuclease PI and alkaline phosphatase, and then analyzed on a HPLC system equipped with an Acclaim 120, CIS, 5 ⁇ Analytical column (4.6x150 mm) (Thermo Scientific, Sunnyvale, CA) e!uted with buffer A (5 mM ammonia acetate) and buffer B (60% acetonitrile, 0.01% TFA, 5 mM ammonia acetate) with a flow rate of 1 ml min "1 at room temperature.
• the detection wave length was set at 260 nm. Quantification of Cellular m6A
• 1 g of mRNA was digested by nuclease PI (2 U) in 40 ⁇ of buffer containing 25 mM of NaCI, and 2.5 mM of ZnCI 2 at 37 ?C for 1 h, followed by the addition of N H4HCO3 (1 M, 3 ⁇ ) and alkaline phosphatase (0.5 U). After an additional incubation at 37°C for 1 h, the solution was diluted 5 times, and 10 ⁇ of the solution was injected into LC-MS/MS.
• the nucleosides were separated by reverse phase ultra-performance liquid chromatography on a CIS column, with online mass spectrometry detection using Agilent 6410 QQQ tripie-quadrupole LC mass spectrometer in positive electrospray ionization mode.
• the nucleosides were quantified using the nucleoside to base ion mass transitions of 282 to 150 (m6A), and 258 to 136 (A). Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards rsjrinirig at the same batch of samples. The ratio of m6A to A was calculated based on the calculated concentrations.
• Statistical analysis performed included the t-test and calculation of the p-value.
• D E fetal bovine serum
• FBS fetal bovine serum
• OPTI-MEM OPTI-MEM
• Compound 5c possesses significant activity in the 6Hz quantitative model, with a seizure suppressive ED 50 of 18 mg/kg, a toxic ED 50 of 347 mg/kg, giving a safety ratio of toxic EDso seizure suppressive ED 50 of 19.
• seizure suppressive ED 50 of 18 mg/kg
• toxic ED 50 of 347 mg/kg
• safety ratio of toxic EDso seizure suppressive ED 50 of 19.
• G2 is the signal from 3c treated (25u ) HeLa cells
• Gl is the signal from untreated control HeLa ceils.
• Gao X Shin YH, Li M, Wang F, Tong Q, Zhang P.
• the fat mass and obesity associated gene FTO functions in the brain to regulate postnatal growth in mice.
• a commonly carried allele of the obesity- related FTO gene is associated with reduced brain volume in the healthy elderly. Proc Natl Acad Sci U S A 2010; 107:8404-9.
• N6-methyladenosine in nuclear RIMA is a major substrate of the obesity-associated FTO.

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