WO2012037105A1 - Procédés de traitement, d'amélioration ou de prévention de troubles et de maladies neuronaux induits par le stress - Google Patents

Procédés de traitement, d'amélioration ou de prévention de troubles et de maladies neuronaux induits par le stress Download PDF

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Publication number
WO2012037105A1
WO2012037105A1 PCT/US2011/051369 US2011051369W WO2012037105A1 WO 2012037105 A1 WO2012037105 A1 WO 2012037105A1 US 2011051369 W US2011051369 W US 2011051369W WO 2012037105 A1 WO2012037105 A1 WO 2012037105A1
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alkyl
aryl
cycloalkyl
heterocyclyl
alkenyl
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PCT/US2011/051369
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English (en)
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Andrew R. Marks
Michael L. Shelanski
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The Trustees Of Columbia University In The City Of New York
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Publication of WO2012037105A1 publication Critical patent/WO2012037105A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/554—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one sulfur as ring hetero atoms, e.g. clothiapine, diltiazem

Definitions

  • the present invention relates to compositions and methods of treating, ameliorating or preventing a stress-induced cognitive dysfunction.
  • RyRs are channels in the sarcoplasmic reticulum (SR) that open and close to regulate the release of Ca 2+ from the SR into the intracellular cytoplasm of the cell.
  • the "open probability" (Po) of a RyR refers to the likelihood that the RyR channel is open at any given moment, and therefore capable of releasing Ca 2+ into the cytoplasm from the SR.
  • RyRl is found predominantly in skeletal muscle as well as other tissues
  • RyR2 is found predominantly in the heart as well as other tissues
  • RyR3 is found in the brain as well as other tissues.
  • the RyR channels are formed by four RyR polypeptides in association with four FK506 binding proteins (FKBPs), specifically FKBP12 (calstabinl) and FKBP12.6 (calstabin2).
  • FKBPs FK506 binding proteins
  • Calstabinl binds to RyRl
  • calstabin2 binds to RyR2
  • calstabinl binds to RyR3.
  • the FKBP proteins calstabinl and calstabin2 bind to the RyR channel (one molecule per RyR subunit), stabilize RyR-channel functioning, and facilitate coupled gating between neighboring RyR channels, thereby preventing abnormal activation of the channel during the channel's closed state.
  • cAMP activates Protein Kinase A (PKA) phosphorylation of RyRl and RyR2 isoforms.
  • PKA Protein Kinase A
  • RyRs function as macromolecular signaling complexes that integrate cAMP and Ca 2+ signaling.
  • RyR dysfunction and pathologic intracellular Ca 2+ leak occur during intense and/or chronic beta-adrenergic stimulation due to sympathetic hyperactivity.
  • Substance abuse also known as drug abuse, refers to a maladaptive pattern of use of a substance that is not considered dependent. Substance abuse refers to taking a psychoactive drug or performance enhancing drug for a non-therapeutic or non-medical effect. Some of the drugs most often associated with this term include alcohol, amphetamines, barbiturates, benzodiazepines (particularly temazepam, nimetazepam, and flunitrazepam), cocaine, methaqualone, and opioids. Use of these drugs may lead to criminal penalty in addition to possible physical, social, and psychological harm, both strongly depending on local jurisdiction. Alcohol dependence is a psychiatric diagnosis (a mental illness) describing an entity in which an individual uses alcohol despite significant areas of dysfunction, evidence of physical dependence, and/or related hardship.
  • compositions and methods useful for the treatment and/or prevention of such disceases and disorders involve modulation of the function of ryanodine receptors.
  • the present invention provides, inter alia, compositions and methods useful for treating, ameliorating or preventing a stress-induced cognitive dysfunction associated with leaky ryanodine receptor (RyR) calcium release channels in neuronal cells in a subject in need thereof.
  • RyR leaky ryanodine receptor
  • the present invention provides a method for treating, ameliorating or preventing a disease or disorder associated with leaky ryanodine receptor (RyR) calcium release channels in neuronal cells by administering certain benzothiazepine, benzoxazepine, benzodiazepine and benzazepine compounds.
  • Rost leaky ryanodine receptor
  • the present invention provides a method of treating, ameliorating or preventing a disease or disorder associated with leaky RyR calcium release channels in neuronal cells in a subject in need thereof, comprising administering to the subject a therapeutically or prophylactically effective amount of a compound of Formula I, I-a', I-a, I- b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1 , 1-1, 1-m, I-n, I-o, I-p, I-a-1, 1-b-1, 1-c-1, 1-d-1, 1-e- 1, I-f- 1 , 1-g-1, I-h- 1 , 1-i-1, or Formula II, or enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes, metabolites, or pro-drugs thereof, or any combination thereof.
  • the structures of these Formulae are provided
  • the present invention provides a method of treating, ameliorating or preventing a disease or disorder associated with leaky RyR calcium release channels in neuronal cells in a subject in need thereof, comprising administering to the subject a therapeutically or prophylactically effective amount of a compound represented by the structure of Formula I-o as disclosed herein.
  • the present invention provides a method of treating, ameliorating or preventing a disease or disorder associated with leaky RyR calcium release channels in neuronal cells in a subject in need thereof, comprising administering to the subject a therapeutically or prophylactically effective amount of the compound SI 07 represented b the structure
  • a preferred salt is the hydrochloride salt (S107-HC1).
  • the present invention provides a method for treating or ameliorating substance abuse or stress-induced cognitive dysfunction associated with substance abuse in a subject in need thereof, comprising administering to the subject a therapeutically or prophylactically effective amount of a compound of Formula I, I-a', I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, 1-1, 1-m, I-n, I-o, I-p, I-a-1, 1-b-1, 1-c-1, 1-d-1, 1-e-1, I-f- 1 , 1-g-1, 1-h-1 , 1-i-1, or Formula II, or enantiomers, diastereomers, tautomers,
  • the substance abuse is selected from the group consisting of alcohol dependence and drug abuse, preferably stress induced by drug abuse.
  • the present invention provides a method of treating, ameliorating or preventing alcohol dependence, drug abuse or stress induced by alcohol dependence or drug abuse in a subject in need thereof, comprising administering to the subject a therapeutically or prophylactically effective amount of a compound of Formula I, I-a', I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, 1-1, 1-m, I-n, I-o, I-p, I-a-1, 1-b-1, 1-c-1, I-d-1, 1-e-1, I-f- 1 , 1-g-1, 1-h-1 , 1-i-1, or Formula II, or enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes, metabolites, or pro-drugs thereof, or any combination thereof.
  • the compounds of the invention are administered to the subject at a dose sufficient to restore or enhance binding of calstabin to RyR by increasing the affinity with which calstabin binds to RyR, or by decreasing dissociation of calstabin from RyR, or by increasing binding of calstabin to RyR.
  • the compounds of the invention are administered to a human subject at a dose of from about 0.01 mg/kg/day to about 20 mg/kg/day, or more preferably still, at a dose of from about 0.05 mg/kg/day to about 1 mg/kg/day.
  • Other suitable dose ranges are provided in the Detailed Description and Examples.
  • one of skill in the art can select other suitable doses for administration.
  • the compound administered is selected from the group consisting of SI, S2, S3, S4, S5, S6, S7, S9, SI 1, S12, SI 3, S14, SI 9, S20, S22, S23, S24, S25, S26, S27, S36, S37, S38, S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S66, S67, S68, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, S100, S101, S
  • the present invention provides compounds of Formula I for use in treating substance abuse and associated stress-induced cognitive dysfunction associated with leaky ryanodine receptor (RyR) calcium release channels in neuronal cells in a subject in need thereof.
  • the compound of Formula I is represented by the structure provided in the Detailed Description that follows.
  • the present invention provides compounds of Formula I for use in preparing a medicament for treating substance abuse and associated stress-induced cognitive dysfunction associated with leaky ryanodine receptor (RyR) calcium release channels in neuronal cells in a subject in need thereof.
  • RyR leaky ryanodine receptor
  • the compound of the invention is administered in an amount that prevents further deterioration of the neuronal cells.
  • Figures 1A-E are bar graphs that show the dose response to SI 07 treatment.
  • Figure 2 includes immunoblots that show SI 07 protection against ⁇ .
  • Figures 3A-C are graphs showing memory and cognitive function in murine model of Alzheimer's tested in the Radial Arm Water Maze (RAWM).
  • RAWM Radial Arm Water Maze
  • Figures 4A and B are bar graphs showing that SI 07 improves fear conditioning in Alzheimer's mouse model.
  • Figures 5A and B are immunoblots that show stress-induced PKA phosphorylation of
  • Figure 6A is a schematic representation of an elevated plus maze for testing mice
  • Figures 6B and 6C are bar graphs that show the performance of mice in the maze.
  • Figure 7 includes immnuoblots and bar graphs that show SI 07 prevents calstabin2 depletion from PKA hyperphosphorylated RyR2 in the brain of isoproterenol treated mice in vivo.
  • Figure 8A is a schematic representation of an elevated plus maze for testing mice
  • Figures 8B-E are bar graphs that show performance of mice in the maze.
  • Figure 9A is a schematic representation of a recognition trial for testing mice
  • Figures 9B-D are bar graphs that show performance of mice in the trial.
  • Figures lOA-C are photomicrographs that show laser confocal imaging of a voltage- dependent Ca 2+ change in the mouse dentate gyrus of a brain slice.
  • Figure 11 shows RyR2RS/WT single-channel traces and histograms from brain vesicles of mice.
  • Figures 12A-H show RyR2RS/WT single-channel traces from mice that were fed ethanol.
  • Figure 13 is a bar graph that shows that ethanol and H 2 0 2 together activate leak in RyRl channels.
  • Figures 14A-D are graphs that show maze testing results.
  • Figures 15A-C are graphs that show cognitive dysfunction of mutant mice in maze testing.
  • Figure 16 includes photomicrographs that show expression of RyRl and RyR2 in different regions of C57BL/6 adult male mouse brain.
  • Figures 17A and B are immunoblots that show whole brain RyR2 biochemistry in wildtype and mutant mice with or without SI 07 treatment.
  • Figures 18A and B include immnuoblots and bar graphs that show phosphorylation of CREB, ERK and CaMKII in wildtype and mutant mice brain cells under stress or without stress.
  • Figures 19A-C are graphs showing that SI 07 restores the neuronal long-term potentiation in stressed WT mice but not stressed knock-in mice with leaky RyR2 channels.
  • Figures 20A-D are bar graphs and immunoblots showing abnormal dentric spine morphology in knock-in mice with leaky RyR2 channels.
  • Figures 21 A and B are photomicrographs that show neuron calcium imaging and calcium signaling in wildtype brain.
  • Figures 22A and B are immunoblots showing that abnormal ryanodine receptor activity is associated with neuronal degenerative disorders.
  • RyCal compounds refers to compounds of the general Formula I, I-a', I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, 1-1, 1-m, I-n, I-o, I-p, or Formula II, as provided by the invention, and herein referred to as "compound(s) of the invention".
  • the compounds of the invention are referred using a numerical naming system, with compound numbers 1 to 477 provided herein. These numbered compounds are referred to using either the prefix “S” or “H”. Thus, the first numbered compound is referred to either as “SI”, the second numbered compound is referred to as “S2”, the third numbered compound is referred to as either “S3”, and so on.
  • SI the first numbered compound
  • S2 the second numbered compound
  • S3 the third numbered compound
  • alkyl refers to a linear or branched, saturated hydrocarbon and preferably one having from 1 to 6 carbon atoms.
  • Representative alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, and neohexyl.
  • C1-C4 alkyl refers to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, and isobutyl.
  • alkenyl refers to a linear or branched hydrocarbon and preferably one having from 2 to 6 carbon atoms and having at least one carbon-carbon double bond. In one embodiment, the alkenyl has one or two double bonds.
  • the alkenyl moiety may exist in the E or Z conformation and the compounds of the present invention include both conformations.
  • alkynyl refers to a linear or branched hydrocarbon and preferably one having from 2 to 6 carbon atoms and having at least one carbon-carbon triple bond.
  • aryl refers to an aromatic group and preferably one containing 1 to 3 aromatic rings, either fused or linked.
  • cyclic group as used herein includes a cycloalkyl group and a heterocyclic group.
  • cycloalkyl group refers to a three- to seven-membered saturated or partially unsaturated carbon ring. Any suitable ring position of the cycloalkyl group may be covalently linked to the defined chemical structure.
  • Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • halogen refers to fluorine, chlorine, bromine, and iodine.
  • heterocyclic group or “heterocyclic” or “heterocyclyl” or “heterocyclo” as used herein refers to fully saturated, or partially or fully unsaturated, including aromatic (i.e., “heteroaryl”) cyclic groups (for example, 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 16 membered tricyclic ring systems) which have at least one heteroatom in at least one carbon atom-containing ring.
  • aromatic i.e., "heteroaryl”
  • Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3, or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.
  • the heterocyclic group may be attached to the remainder of the molecule at any heteroatom or carbon atom of the ring or ring system.
  • Non-limiting examples of heterocyclic groups include, but are not limited to, azepanyl, azetidinyl, aziridinyl, dioxolanyl, furanyl, furazanyl, homo piperazinyl,
  • pyridooxazolyl pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiomorpholinyl, thiophenyl, triazinyl, and triazolyl.
  • bicyclic heterocyclic groups include indolyl, isoindolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, quinuclidinyl, quinolinyl,
  • tetrahydroisoquinolinyl isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, benzofurazanyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] or furo[2,3- b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo- quinazolinyl), triazinylazepinyl, tetrahydroquinolinyl and the like.
  • tricyclic heterocyclic groups include carbazolyl, benzidolyl,
  • phenyl refers to a substituted or unsubstituted phenyl group.
  • alkyl alkenyl
  • alkynyl alkynyl
  • aryl acyl
  • phenyl cyclic group
  • cycloalkyl heterocyclyl
  • heterocyclo heterocyclo
  • heterocycle may further be optionally substituted with one or more substituents.
  • R * is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, alkylaryl, heteroaryl, heterocycle, or aryl
  • R , R c and R d are independently hydrogen, alkyl, cycloalkyl, alkylaryl, heteroaryl, heterocycle, aryl, or said R b and R c together with the N to which they are bonded optionally form a heterocycle
  • R e is alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkylaryl, heteroaryl, heterocycle, or aryl.
  • groups such as alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, alkylaryl, heteroaryl, heterocycle and aryl can themselves be optionally substituted.
  • Non-limiting examples of substituents may further optionally include at least one labeling group, such as a fluorescent, a bio luminescent, a chemiluminescent, a colorimetric or a radioactive labeling group.
  • a fluorescent labeling group can be selected from bodipy, dansyl, fluorescein, rhodamine, Texas red, cyanine dyes, pyrene, coumarins, Cascade BlueTM, Pacific Blue, Marina Blue, Oregon Green, 4',6-Diamidino-2-phenylindole (DAPI), indopyra dyes, lucifer yellow, propidium iodide, porphyrins, arginine, and variants and derivatives thereof.
  • DAPI 6-Diamidino-2-phenylindole
  • SI 18 of the present invention contains a labeling group BODIPY, which is a family of fluorophores based on the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene moiety.
  • BODIPY is a family of fluorophores based on the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene moiety.
  • quaternary nitrogen refers to a tetravalent positively charged nitrogen atom including, for example, the positively charged nitrogen in a tetraalkylammonium group (e.g., tetramethylammonium, N-methylpyridinium), the positively charged nitrogen in protonated ammonium species (e.g., trimethyl-hydroammonium, N-hydropyridinium), the positively charged nitrogen in amine N-oxides (e.g., N-methyl-morpholine-N-oxide, pyridine-N-oxide), and the positively charged nitrogen in an N-amino-ammonium group (e.g., N- aminopyridinium) .
  • a tetraalkylammonium group e.g., tetramethylammonium, N-methylpyridinium
  • protonated ammonium species e.g., trimethyl-hydroammonium, N-hydropyridinium
  • benzothiazepine ring of compounds of the present invention may optionally be a quaternary nitrogen.
  • Non- limiting examples include SI 13 and SI 19.
  • the compounds described herein may exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present invention.
  • prodrug denotes a compound that, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield compounds of the present invention.
  • compound(s) of the invention means a compound of Formula I, I-a', I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, 1-1, 1-m, I-n, I-o, I-p, or Formula II, or any of the specific chemical compounds described herein, and salts, hydrates, complexes, metabolites, prodrugs and solvates thereof, or any combination thereof, such as may be used for the treatment or prevention of stress related disorders or diseases.
  • a “pharmaceutical composition” refers to a mixture of one or more of the compounds described herein, or pharmaceutically acceptable salts, hydrates or pro-drugs thereof, with other chemical components, such as physiologically acceptable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • pro-drug refers to an agent which is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they are easier to administer than the parent drug. They are bioavailable, for instance, by oral administration whereas the parent drug is not. The pro-drug also has improved solubility in pharmaceutical compositions over the parent drug.
  • the compound carries protective groups which are split off by hydrolysis in body fluids, e.g., in the bloodstream, thus releasing active compound or is oxidized or reduced in body fluids to release the compound.
  • a compound of the present invention also can be formulated as a pharmaceutically acceptable salt, e.g., acid addition salt, and complexes thereof.
  • a pharmaceutically acceptable salt e.g., acid addition salt, and complexes thereof.
  • the preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of the agent without preventing its physiological effect. Examples of useful alterations in physical properties include, but are not limited to, lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.
  • salts means a salt that is suitable for, or compatible with, the treatment of a patient or a subject such as a human patient.
  • the salts can be any non-toxic organic or inorganic salt of any of the compounds represented by Formula I, I-a', I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1 , 1-1, 1-m, I-n, I-o, I-p or any of the specific compounds described herein, or any of their intermediates.
  • Illustrative salt-forming ions include, but are not limited to, ammonium (NH 4 ), calcium (Ca” T ), iron (Fe iT and Fe ), magnesium (Mg 2+ ), potassium (K + ), pyridinium (CSHSN T), quaternary ammonium (NP + ), sodium (Na + ), acetate, carbonate, chloride, bromide, citrate, cyanide, hydroxide, nitrate, nitrite, oxide, phosphate, sulfate, maleate, fumarate, lactate, tartrate, gluconate, besylate, and valproate.
  • NH 4 ammonium
  • Ca calcium
  • Fe iT and Fe iron
  • Mg 2+ magnesium
  • K + potassium
  • CSHSN T pyridinium
  • NP + quaternary ammonium
  • Na + sodium (Na + ), acetate, carbonate, chloride, bromide, citrate, cyanide, hydro
  • Illustrative inorganic acids that form suitable salts include, but are not limited to, hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate.
  • Illustrative organic acids that form suitable acid addition salts include, but are not limited to, mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids.
  • Either mono or di-acid salts can be formed, and such salts exist in either a hydrated, solvated or substantially anhydrous form.
  • the acid addition salts of compounds of Formula I, I-a', I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1 , 1-1, 1-m, I-n, I-o, and I-p are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms.
  • the selection of an appropriate salt can be performed by one skilled in the art. For example, one can select salts in reference to "Handbook of
  • regioisomers, configurational isomers, conformers or diasteroisomeric forms all such forms and various mixtures thereof are included in the scope of Formula I, I-a', I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, 1-1, 1-m, I-n, I-o, I-p, or Formula II. It is possible to isolate individual isomers using known separation and purification methods, if desired. For example, when a compound of the present invention is a racemate, the racemate can be separated into the (S)-compound and (R)-compound by optical resolution.
  • solvate as used herein means a compound of Formula I, I-a', I-a, I-b, I-c, I- d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, 1-1, 1-m, I-n, I-o, I-p, or Formula II, or a pharmaceutically acceptable salt thereof, wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate.”
  • an “effective amount,” “sufficient amount,” “therapeutically effective amount,” or “prophylactically effective amount” of an agent or compounds, as used herein, refer to amounts sufficient to effect the beneficial or desired results, including clinical results and, as such, the actual “amount” intended will depend upon the context in which it is being applied, such as whether the desired clinical outcome is prevention or treatment.
  • the term “effective amount” also includes that amount of the compound of Formula I, I-a', I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, 1-1, 1-m, I-n, I-o, I-p, or Formula II, which is
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment. Unless otherwise stated, the term “treatment” should be construed as encompassing preventive and therapeutic methods.
  • to prevent means that a subject who is predisposed to or at risk of developing a disease or disorder, due to family genetic background or age, is not expected to develop such disease or disorder as a result of administering the rycal compound to the subject.
  • to ameliorate means the administering the rycal compound to a subject after the onset of the disease or disorder slows down the development of such disease or disorder.
  • animal refers to all members of the animal kingdom including, but not limited to, mammals, animals (e.g., cats, dogs, horses, etc.) and humans.
  • All stereoisomers of the compounds of the present invention are contemplated within the scope of this invention.
  • Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers (e.g., as a pure or substantially pure optical isomer having a specified activity), or may be admixed, for example, as racemates or with all other, or other selected,
  • the chiral centers of the present invention may have the S or R configuration as defined by the IUPAC 1974 Recommendations.
  • the racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography.
  • the individual optical isomers can be obtained from the racemates by any suitable method, including without limitation, conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.
  • Compounds of the present invention are, subsequent to their preparation, preferably isolated and purified to obtain a composition containing an amount by weight equal to or greater than 95% and preferably more than 99% of the compound ("substantially pure” compound), which is then used or formulated as described herein. Such “substantially pure” compounds of the present invention are also contemplated herein as part of the present invention.
  • cognitive function and “cognitive process” as used herein, include the mental processes of attention, learning and memory, perception, language skills, problem solving skills, and other type of cognitive function known to those of skill in the art.
  • cognitive disorder refers to situations in which processes are disrupted or abnormal.
  • cognitive disorder encompasses all of the cognitive disorders described below and also all other cognitive disorders known to those of skill in the art. Types of cognitive disorders that are within the scope of the invention include, but are not limited to, dementias, deli ium and amnesias.
  • dementia refres to decline in cognitive function due to damage or disease in the brain or central nervous system beyond that which might be expected from normal aging. Dementias typically affect cognitive functions such as learning, memory, attention, language skills, and problem solving skills. Types and causes of dementia include, but are not limited to, Alzheimer's disease, vascular dementia (also known as multiinfarct dementia), Binswanger's disease, dementia with Lewy bodies (DLB), alcohol-induced persisting dementia, frontotemporal lobar degenerations (FTLD), Pick's disease,
  • frontotemporal dementia or frontal variant FTLD
  • semantic dementia or temporal variant FTLD
  • progressive non-fluent aphasia Creutzfeldt-lakob disease
  • Huntington's disease Parkinson's di and AIDS dementia complex.
  • any combination of all the recited substitutents are within the scope of the invention.
  • these compounds are related in that they regulate the function of the ryanodine receptors, in particular, the interaction between the ryandine receptor and its binding protein calstabin, it is expected that they have similar therapeutical or prophylactical functions, and there is no reason to believe otherwise.
  • all the compounds to be administered in the method of the invention have the same bicyclic structure. It is to be understood that the possible substitutents as recited herein do not disrupt the bicyclic backbone structure of the compounds of the invention, although additional ring structures can be formed involving atoms not part of the bicyclic backbone.
  • the compounds to be administered in the method of the invention also includes the compounds described in published PCT applications WO 07/024717, WO 08/144483 and WO 09/026444, the entire contents of each of which are hereby incorporated by reference, and included in Appendices I-III.
  • mice subjected to chronic restraint stress showed significantly increased levels of RyPvl and RyR2 PKA phosphorylation in the brain, increased RyRl and RyR2 channel activity, and altered behavior and impaired cognitive function.
  • Chronic RyR PKA Chronic RyR PKA
  • intracellular Ca 2+ leak represents a key pathology that impairs memory storage and alters signaling in the brain required for normal behavior (e.g., threat avoidance) and contributes to post-traumatic stress disorder (PTSD), alcohol dependence, and Alzheimer's disease.
  • PTSD post-traumatic stress disorder
  • the present invention uses an entirely novel and innovative approach of preventing RyR mediated intracellular Ca 2+ leak with a 1 ,4-benzothiazepine compound (rycal). Consistently, the inventors have previously shown that chronic maximal stress results in permanent activation of the sympathetic nervous system causing RyR PKA hyperphosphorylation and intracellular Ca 2+ leak. In the brain, intracellular Ca 2+ leak gradually causes cognitive dysfunction, impaired memory acquisition, and behavioral abnormalities.
  • a drug like a rycal which fixes the SR Ca 2+ leak by binding calstabin to the channel and stabilizing the closed state even during stress therefore helps to prevent cognitive dysfunction, and represents a novel and unique approach to improving cognitive function in the setting of chronic stress.
  • the pharmacotherapy is unique since it targets a central mechanism and potentially prevents toxic effects of intracellular Ca 2+ leak. Moreover, the molecular mechanism of this pharmocotherapy is revolutionary, since it is a novel approach to treat a specific defect contributing to cognitive dysfunction and since the mechanism of rycal action is stabilization of normal RyR channel closure by increasing the calstabin binding affinity, which is distinct from historical approaches used to treat cognitive disorders.
  • a key aspect of the present invention is to prevent deleterious effects of stress exposure without blocking the physiological activities of neuronal ion channels required for normal brain function and survival.
  • amyloid- ⁇ at 300 nM was chosen because this is the highest tolerated dose that does not cause apoptosis. There was no cell death with 24 hr of amyloid- ⁇ (" ⁇ ") treatment when observing synaptic changes (see, e.g., Fig. 1C).
  • the inventors have also developed a novel RyR-targeted pharmacological strategy that stabilizes the RyRl and RyR2 closed state in PKA hyperphosphorylated channels and prevents intracellular Ca 2+ leak induced pathophysiology in vivo.
  • a novel RyR-targeted pharmacological strategy that stabilizes the RyRl and RyR2 closed state in PKA hyperphosphorylated channels and prevents intracellular Ca 2+ leak induced pathophysiology in vivo.
  • compositions described herein are administered therapeutically or prophylactically to subjects who are suffering from, or at risk of developing stress induced neuronal diseases or disorders, due to family genetic background or age.
  • a subject may be any animal that is suffering from, or at risk of developing stress induced neuronal diseases or disorders.
  • the subject is a mammal.
  • mammals that may be treated using the methods and compositions of the invention include, but are not limited to, primates, rodents, ovine species, bovine species, porcine species, equine species, feline species and canine species.
  • the subjects are human.
  • the "subjects" of the present invention may also be in vitro or in vivo systems, including, without limitation, isolated or cultured cells or tissues, in vitro assay systems.
  • compositions for preparing and/or preparing the compounds described herein may be formulated into compositions for preparing and/or preparing the compounds described herein.
  • compositions comprise one or more of the 1 ,4-benzothiazepine compounds described herein (such as the compounds of Formula I, I-a', I-a, I-b, I-c, I-d, I-e, I- f, I-g, I-h, I-i, I-j, I-k, I-k-1, 1-1, 1-m, I-n, I-o, I-p, or Formula II), in admixture with a pharmaceutically acceptable diluent and/or carrier and optionally one or more other pharmarceutically acceptable additives.
  • a pharmaceutically acceptable diluent and/or carrier optionally one or more other pharmarceutically acceptable additives.
  • compositions suitable for administration to subjects, such as human subjects, for example using the teaching a standard text such as Remington's Pharmaceutical Sciences, 18th ed, (Mack Publishing Company: Easton, Pa., 1990), pp. 1635-36), and by taking into account the selected route of delivery.
  • diluents and/or carriers and/or other additives that may be included in the compostions of the invention include, but are not limited to, water, glycols, oils, alcohols, aqueous solvents, organic solvents, DMSO, saline solutions, physiological buffer solutions, peptide carriers, starches, sugars, preservatives, antioxidants, coloring agents, pH buffering agents, granulating agents, lubricants, binders, disintegrating agents, emulsifiers, binders, excipients, extenders, glidants, solubilizers, stabilizers, surface active agents, suspending agents, tonicity agents, viscosity-altering agents, carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate.
  • diluents and/or carriers and/or other additives used can be varied taking into account the nature of the active agents used (for example the solubility and stability of the active agents), the route of delivery (e.g. oral, parenteral, etc.), whether the agents are to be delivered over an extended period (such as from a controlled- release capsule), whether the agents are to be co-administered with other agents, and various other factors.
  • the route of delivery e.g. oral, parenteral, etc.
  • an extended period such as from a controlled- release capsule
  • agents are to be co-administered with other agents
  • the compounds of Formula I, I- a', I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, 1-1, 1-m, I-n, I-o, I-p, or Formula II may be administered to the subject (or contacted with cells of the subject) in an amount effective to treat and/or prevent stress induced neuronal diseases or disorders, and/or in an amount effective to reduce calcium "leak" through the RyR channel, and/or in an amount effective to reduce the calcium current through the RyR channel, and/or in an amount effective to stabilize gating of the RyR channel, and/or in amount effective to increase the binding of calstabin to the RyR complex in the subject, and/or in amount effective to reverse a malfunction of a RyR channel in the subject, particularly in the neuronal cells of the subject.
  • an effective amount of the agents of the invention to be administered to a subject taking into account whether the agent is being used prophylactically or therapeutically, and taking into account other factors such as the age, weight and sex of the subject, any other drugs that the subject may be taking, any allergies or contraindications that the subject may have, and the like.
  • an effective amount can be determined by the skilled artisan using known procedures, including analysis of titration curves established in vitro or in vivo.
  • the desired subject is a human
  • one of skill in the art can determine the effective dose from performing pilot experiments in suitable animal model species and scaling the doses up or down depending on the subjects weight etc.
  • Effective amounts can also be determined by performing clinical trials in individuals of the same species as the subject, for example starting at a low dose and gradually increasing the dose and monitoring the effects on stress induced neuronal diseases or disorders. Appropriate dosing regimens can also be determined by one of skill in the art without undue experimentation, in order to determine, for example, whether to administer the agent in one single dose or in multiple doses, and in the case of multiple doses, to determine an effective interval between doses.
  • an effective amount of the compounds of the invention to administer to a subject ranges from about 0.01 mg/kg/day to about 1000 mg/kg/day, and/or is an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml.
  • the amount of compounds from the invention ranges from about 5 mg/kg/day to about 20 mg/kg/day. In another embodiment, from about 10 mg/kg/day to about 20 mg/kg/day is administered. In another embodiment, from about 0.01 mg/kg/day to about 10 mg/kg/day is administered. In another embodiment, from about 0.01 mg/kg/day to about 5 mg/kg/day is administered.
  • the amount of compounds from the invention ranges from about 50 mg/kg/day to about 500 mg/kg/day. In another preferred embodiment, the amount of compounds from the invention ranges from about 50 mg/kg/day to about 100 mg/kg/day.
  • compositions described herein may be administered to a subject by any suitable method that allows the agent to exert its effect on the subject in vivo.
  • the compositions may be administered to the subject by known procedures including, but not limitated to, by oral administration, sublingual or buccal administration, parenteral administration, transdermal administration, via inhalation, via nasal delivery, vaginally, rectally, and intramuscularly.
  • the compounds of the invention may be administered parenterally, or by epifascial, intracapsular, intracutaneous, subcutaneous, intradermal, intrathecal, intramuscular, intraperitoneal, intrasternal, intravascular, intravenous, parenchymatous, or sublingual delivery.
  • Delivery may be by injection, infusion, catheter delivery, or some other means, such as by tablet or spray.
  • the agent is adiminstered to the subject by way of delivery directly to the heart tissue, such as by way of a catheter inserted into, or in the proximity of the subject's heart, or by using delivery vehicles capable of targeting the drug to the heart.
  • the compounds of the invention may be conjugated to or administered in conjunction with an agent that is targeted to the heart, such as an antibody or antibody fragment.
  • a formulation of the compounds of the invention may be presented as capsules, tablets, powders, granules, or as a suspension or solution.
  • the formulation may contain conventional additives, such as lactose, mannitol, cornstarch or potato starch, binders, crystalline cellulose, cellulose derivatives, acacia, cornstarch, gelatins, disintegrators, potato starch, sodium carboxymethylcellulose, dibasic calcium phosphate, anhydrous or sodium starch glycolate, lubricants, and/or or magnesium stearate.
  • the compounds of the invention may be combined with a sterile aqueous solution that is isotonic with the blood of the subject.
  • a sterile aqueous solution that is isotonic with the blood of the subject.
  • Such a formulation may be prepared by dissolving the active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering the solution sterile.
  • the formulation may be presented in unit or multi-dose containers, such as sealed ampules or vials.
  • the formulation may be delivered by injection, infusion, or other means known in the art.
  • the compounds of the invention may be combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone and the like, which increase the permeability of the skin to the compounds of the invention and permit the compounds to penetrate through the skin and into the bloodstream.
  • skin penetration enhancers such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone and the like, which increase the permeability of the skin to the compounds of the invention and permit the compounds to penetrate through the skin and into the bloodstream.
  • compositions also may be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which are dissolved in a solvent, such as methylene chloride, evaporated to the desired viscosity and then applied to backing material to provide a patch.
  • a polymeric substance such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like
  • a solvent such as methylene chloride
  • the composition is in unit dose form such as a tablet, capsule or single-dose injection or infusion vial.
  • the agents described herein may be used in combination with other agents useful for the treatment of stress induced neuronal diseases or disorders or with other agents that ameliorate the effect of certain risk factors for stress induced neuronal diseases or disorders.
  • the agents of the invention may be delivered to a subject as part of a composition containing one or more additional active agents.
  • the agents of the invention may be delivered to a subject in a composition or formulation containing only that active agent, while one or more other agents useful for the treatment and/or prevention of stress induced neuronal diseases or disorders may also be administered to the subject in one or more separate compositions or formulations.
  • agents of the invention and the other agents useful for the treatment and/or prevention of stress induced neuronal diseases or disorders may be administered to the subject at the same time, or at different times.
  • the agents of the invention and the other agents may be administered within minutes, hours, days, weeks, or months of each other, for example as part of the overall treatment regimen of a subject.
  • Agents of the invention useful for treating and/or preventing stress induced neuronal diseases or disorders may be used in combination with the other agents that include, but are not limited to, ⁇ -adrenergic blockers, calcium channel blockers and anti-arrhythmic drugs.
  • the present invention is directed to methods for identifying additional compounds that may be useful for the treatment and/or prevention of stress induced neuronal diseases or disorders.
  • Such methods may be based on, inter alia, identifying compounds that increase binding of calstabins to RyRs, and/or decrease the calcium current through RyR channels, and the like. Examples of suitable assays and screening methods that may be used to identify new compounds that possess these properties such that they may be useful for the treatment and/or prevention of stress induced neuronal diseases or disorders are described in U.S.
  • the present invention encompasses compounds useful for the treatment and/or prevention of stress-induced cognitive dysfunction, and methods of treatment and/or prevention comprising administration of such compounds, or compositions containing such compounds, to subjects who are suffereing from, or who are at risk of developing stress- induced cognitive dysfunction.
  • the compounds of the invention indirectly decrease the open probability of RyR when examined under conditions that simulate diastole, by inhibiting the depletion of the stabilizing subunit calstabin2 from the RyR2 complex and thereby stabilizing the closed state of the channel, particularly PKA-phosphorylated, and/or nityrosylated, and/or oxidized RyR, and thereby decrease the Ca 2+ current through such channels under resting conditions when muscles are relaxed.
  • the compounds of the invention exert this effect, at least in part, by increasing the affinity with which calstabin proteins bind to RyRs, and/or by inhibiting a decrease in binding of calstabins to RyRs, and/or by inhibiting dissociation of calstabins from RyRs, particularly PKA phosphorylated RyRs.
  • the compounds of the invention decrease the open probability of RyR and decrease the "leak" of Ca 2+ through such channels by stabilizing the closed state of the channel without blocking the channel pore.
  • the present invention relates to use of benzothiazepine, benzoxazepine,
  • the present invention provides benzothiazepine, benzoxazepine, benzodiazepine and benzazepine compounds as described by the chemical Formula I, I-a', I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, 1-1, 1-m, I-n, I- o, I-p, I-a-1, I-b- 1 , 1-c-1, I-d- 1 , 1-e-1, 1-f-1, I-g- 1 , 1-h-1, I-i- 1 , or Formula II, as described below.
  • the present invention provides methods for the treatment and/or prevention of stress-induced cognitive dysfunction that comprise administering compounds of Formula I to subjects in need thereof.
  • the present invention provides compositions useful for the treatment and/or prevention of stress-induced cognitive dysfunction that comprise compounds of Formula I.
  • the structure of Formula I is as follows: (Formula I)
  • n 0, 1, or 2;
  • q 0, 1, 2, 3, or 4;
  • Ri is selected from the group consisting of H, oxo, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be optionally substituted;
  • Re is selected from the group consisting of -OR15, -NHNR15R16, -NHOH, -NR15R16, -CH 2 X, acyl, alkenyl, alkyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, alkylaryl, cycloalkyl,
  • cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted
  • R 7 is selected from the group consisting of -OR15, -NR 15 R 16 , -NHNR15R16, -NHOH, -CH 2 X, alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;
  • Rg and R9 independently are selected from the group consisting of OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted;
  • R11, Ri 2 , Ri 3 , and Ri 4 independently are selected from the group consisting of H, OH, NH 2 , -NHNH 2 , -NHOH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl,
  • heterocyclyl, and heterocyclylalkyl may be optionally substituted
  • X is selected from the group consisting of halogen, -CN, -C0 2 Ri 5 , -NR 15 R 16 , Ri 5 and Ri 6 independently are selected from the group consisting of H, acyl, alkenyl, alkoxyl, OH, NH 2 , alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally substituted; and optionally R15 and Ri 6 together with the N to which they are bonded may form a heterocycle which may be substituted;
  • the nitrogen in the benzothiazepine ring may optionally be a quaternary nitrogen; and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes, and prodrugs thereof.
  • Examples of compounds that may be used in conjunction with the invention include, without limitation, SI, S2, S3, S4, S5, S6, S7, S9, SI 1, S12, SI 3, S14, SI 9, S20, S22, S23, S24, S25, S26, S27, S36, S37, S38, S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S66, S67, S68, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, S100, S101,
  • the present invention provides methods and uses which comprise administering compounds of Formula I-a':
  • q 0, 1, 2, 3, or 4;
  • Re is selected from the group consisting of -OR15, -NHNR15R16, -NHOH, -NR15R16, -CH 2 X, acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted;
  • R 7 is selected from the group consisting of H, -OR15, -NR15R16, -NHNR15R16, -NHOH, -CH 2 X, alkyl, akenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
  • heterocyclylalkyl wherein each alkyl, akenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted;
  • Rg and R9 independently are selected from the group consisting of -OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl;
  • each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted;
  • Rii, Ri 2 , Ri 3 , and R14 independently are selected from the group consisting of H, OH, NH 2 , -NHNH 2 , -NHOH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl;
  • Ri 5 and Ri 6 independently are selected from the group consisting of H, acyl, alkenyl, alkoxyl, OH, NH 2 , alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
  • heterocyclylalkyl wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted; and optionally Ri 5 and R 1 ⁇ 2 together with the N to which they are bonded may form a heterocycle which may be substituted or unsubstituted;
  • the nitrogen in the benzothiazepine ring may be optionally a quaternary nitrogen; and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes, and prodrugs thereof.
  • the present invention provides methods and uses which comprise administering compounds of Formula I-a:
  • n 0, 1, or 2;
  • q 0, 1, 2, 3, or 4;
  • Re is selected from the group consisting of -ORi 5 , -NHNR15R16, -NHOH, -NR15R16, -CH 2 X, acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted;
  • R 7 is selected from the group consisting of H, -OR15, -NR 15 R 16 , -NHNR15R16, -NHOH, -CH 2 X, alkyl, akenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
  • heterocyclylalkyl wherein each alkyl, akenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted;
  • R 8 and R 9 independently are selected from the group consisting of -OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl;
  • each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted;
  • R 11 , Ri 2 , Ri 3 , and Ri 4 independently are selected from the group consisting of H, OH, NH 2 , -NHNH 2 , -NHOH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted;
  • Ri5 and Ri 6 independently are selected from the group consisting of H, acyl, alkenyl, alkoxyl, OH, NH 2 , alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
  • heterocyclylalkyl wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted; and optionally R15 and R1 ⁇ 2 together with the N to which they are bonded may form a heterocycle which may be substituted or unsubstituted;
  • the nitrogen in the benzothiazepine ring may be optionally a quaternary nitrogen; and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes, and prodrugs thereof.
  • n 0, 1, or 2.
  • the present invention provides methods and uses which comprise administering compounds of formula I-b:
  • R 2 and n are as defined in compounds of formula I-a above;
  • R is H or OMe, and R" is H.
  • the present invention provides methods and uses which comprise administering compounds formula of I-c:
  • each R, R 7 , q, and n is as defined in compounds of formula I-a above; and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes, metabolites, and pro-drugs thereof.
  • the present invention provides methods and uses which comprise administering compounds of formula I-c, wherein R 7 is selected from the group consisting of -OH, -NR 15 R 16 , alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, akenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted.
  • the present invention provides methods and uses which comprise administering com ounds of formula of I-d:
  • R 7 and n are as defined in compounds of formula I-a above; and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes and pro-drugs thereof.
  • R is H or OMe
  • R" is H.
  • the present invention provides methods and uses which comprise administering compounds of formula I-d, wherein R 7 is selected from the group consisting of -OH, -NR 15 R 16 , alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, akenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted.
  • the present invention provides methods and uses which comprise administering compounds of formula of I-e:
  • each R, R 5 , q and n is as defined compounds of formula I-a above; and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes and pro-drugs thereof.
  • the present invention provides methods and uses which comprise administering compounds of formula I-e, wherein R 5 is selected from the group consisting of-NRi 5 Ri 6 , -(CH 2 ) z NRi 5 Ri 6 , -NHOH, -ORi 5 , -CH 2 X, alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted.
  • the present invention provides methods and uses which comprise administering compounds of formula I-e, wherein R5 is an alkyl substituted by at least one labeling group, such as a fluorescent, a bioluminescent, a chemiluminescent, a colorimetric and a radioactive labeling group.
  • R5 is an alkyl substituted by at least one labeling group, such as a fluorescent, a bioluminescent, a chemiluminescent, a colorimetric and a radioactive labeling group.
  • a fluorescent labeling group can be selected from bodipy, dansyl, fluorescein, rhodamine, Texas red, cyanine dyes, pyrene, coumarins, Cascade BlueTM, Pacific Blue, Marina Blue, Oregon Green, 4',6-Diamidino-2-phenylindole (DAPI), indopyra dyes, lucifer yellow, propidium iodide, porphyrins, arginine, and variants and derivatives thereof.
  • the present invention provides methods and uses which mprise administering compounds of formula of I-f:
  • R 5 and n are as defined in compounds of formula I-a above;
  • R is H or OMe
  • R" is H.
  • a preferred compound of formula I-f is S36, in particular in the form of a sodium salt.
  • the present invention provides methods and uses which comprise administering compounds of formula I-f, wherein -(CH 2 ) z NRi 5 Ri6, selected from the group consisting of-NRi 5 Ri 6 , -NHOH, -OR15, -CH 2 X, alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted.
  • the present invention provides methods and uses which comprise administering compounds of formula of I-g:
  • W is S or O; each R, Ri 5 , Ri 6 , q, and n is as defined in compounds of formula I-a above; and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes and pro-drugs thereof.
  • the present invention provides methods and uses which comprise administering compounds of formula I-g, wherein Ri 5 and Ri 6 independently are selected from the group consisting of H, OH, NH 2 , alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted; and optionally R15 and R1 ⁇ 2 together with the N to which they are bonded may form a heterocycle which may be substituted.
  • Ri 5 and Ri 6 independently are selected from the group consisting of H, OH, NH 2 , alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkyla
  • the present invention provides methods and uses which comprise administering compounds of formula I-g, wherein W is O or S.
  • the present invention provides methods and uses which comprise administering compounds of formula of I-h:
  • W is S or O
  • Ri 5 , Ri 6 and n are as defined in compounds of formula I-a above;
  • R is H or OMe, and R" is H.
  • the present invention provides methods and uses which comprise administering compounds of formula I-h, wherein Ri 5 and Ri 6 independently are selected from the group consisting of H, OH, NH 2 , alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted; and optionally Ri 5 and R 1 ⁇ 2 together with the N to which they are bonded may form a heterocycle which may be substituted.
  • the present invention provides methods and uses which comprise administering compounds of formula I-h, wherein W is O or S.
  • the present invention provides methods and uses which comprise administering compounds of formula of I-i:
  • Rn is selected from the group consisting of -NR15R16, -NHNR15R16, -NHOH, -OR15, -CH 2 X, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl;
  • alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted;
  • each R, q, and n is as defined in compounds of formula I-a above; and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes and pro-drugs thereof.
  • the present invention provides methods and uses which comprise administering compounds of formula I-i, wherein each R is independently selected from the group consisting of H, halogen, -OH, OMe, -NH 2 , -N0 2 , -CN, -CF 3 , -OCF 3 , -N 3 ,
  • n 0, 1, or 2.
  • the present invention provides methods and uses which comprise administering compounds of formula I-i, wherein Rn is -NR 15 R 16 , and -OR 15 .
  • Rn is -OH, -OMe, -NEt, -NHEt, -NHPh, -NH 2 , or
  • the present invention provides methods and uses which comprise administering compounds of formula of I-j :
  • Riv is selected from the group consisting of -NRi 5 Ri 6 , -NHNRi 5 Ri 6 , -NHOH, -ORi 5 , -CH 2 X, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted;
  • n is as defined in compounds of formula I-a;
  • R is H or OMe
  • R" is H.
  • the present invention provides methods and uses which comprise administering compounds of formula I-j, wherein R 17 is -NR15R16 or -OR15.
  • R n is -OH, -OMe, -NEt, -NHEt, -NHPh, -NH 2 , or
  • the present invention provides methods and uses which comprise administering compounds of formula I-k-1:
  • p is any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, with each value of p representing a different embodiment
  • each R, Ri 5 , Ri 6 , q, and n is as defined in compounds of formula I-a above, and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes and pro-drugs thereof.
  • the present invention provides methods and uses which comprise administering mpounds of formula I-k:
  • R and R" are independently selected from the group consisting of H, halogen, -OH,
  • acyl alkyl, alkoxyl, alkylamino, alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; and wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio may be substituted or unsub
  • p is any one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, with each value of p representing a different embodiment; and n is 0, 1 , or 2; and enantiomers, diastereomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, complexes and pro-drugs thereof.
  • the present invention provides methods and uses which comprise administering compounds of formula I-k, wherein R and R" are
  • n is 1
  • n
  • n is 0, and Rig is Ci-C 4 alkyl, such as Me, Et, propyl, and butyl.
  • n is 2, and Rig is pyrrolidine, piperidine, piperazine, or morpholine.
  • m is 3, 4, 5, 5, 7, or 8, and Rig is a fluorescent labeling group selected from bodipy, dansyl, fluorescein, rhodamine, Texas red, cyanine dyes, pyrene, coumarins, Cascade BlueTM, Pacific Blue,
  • R is H, OMe, or C 2 -C 4 alkoxyl;
  • R" is H;
  • n is 0; and
  • Rig is Ci-C 4 alkyl.
  • the present invention provides methods and uses which comprise administering com ounds of formula of I-l:
  • R 6 and n are as defined in compounds of formula I-a;
  • R is H or OMe
  • R" is H.
  • the present invention provides methods and uses which comprise administering compounds of formula I-l, wherein R ⁇ 5 is selected from the group consisting of -NR15R16, -NHNR15R16, -OR15, -NHOH, -CH 2 X, acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted or unsubstituted.
  • Re is -NRi 5 Ri 6 such as -NHPh, pyrrolidine, piperidine, piperazine, morpholine, and the like.
  • R 6 is alkoxyl, such as -O-tBu.
  • the present invention provides methods and uses which comprise administering com ounds of formula I-m:
  • R' and R" are independently selected from the group consisting of H, halogen, -OH,
  • acyl alkyl, alkoxyl, alkylamino, alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; and wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio may be substituted or unsubsti
  • R8, R9 and n are as defined in compounds of formula I-a above; and enantiomers,
  • R is H or OMe
  • R" is H.
  • the present invention provides methods and uses which comprise administering compounds of formula I-m, wherein Rg and R9 are independently alkyl, aryl, -OH, alkoxyl, or alkylamino.
  • R 8 is Ci-C 4 alkyl such as Me, Et, propyl and butyl; and R9 is aryl such as phenyl.
  • the present invention provides methods and uses which comprise administering compounds of formula I-n,
  • R d is CH 2 , or NR a ;
  • Ra is H, -(Ci-C 6 alkyl)-aryl, wherein the aryl is a disubstituted phenyl or a benzo[l,3]dioxo-5-yl group, or an amine protecting group (e.g., a Boc group); and
  • Rb is hydrogen of alkoxy (e.g., methoxy).
  • Representative compounds of Formula I-n include without limitation SlOl, SI 02, S103, S114.
  • the invention provides compounds of Formula I-o:
  • Rs is substituted or unsubstituted -Ci-C 6 alkyl, -(Ci-C 6 alkyl)-phenyl, or -(Ci-C 6 alkyl)-C(0)R b ;
  • R b is -OH or -0-(Ci-C 6 alkyl), and
  • phenyl or substituted alkyl is substituted with one or more of halogen, hydroxyl, -Ci-C 6 alkyl, -0-(Ci-C 6 alkyl), -NH 2 , -NH(Ci-C 6 alkyl), -N(Ci-C 6 alkyl) 2 , cyano, dioxolane.
  • Representative compounds of Formula I-o include without limitation S 107, SI 10, Si l l, S120, and S121.
  • the invention provides compounds of Formula I-p:
  • Rc is -(Ci-Ce alkyl)-NH 2 , -(Ci-C 6 alkyl)-OR f , wherein R f is H or -C(0)-(Ci-C 6 )alkyl, or -(Ci-C 6 alkyl)-NHR g wherein Rg is carboxybenzyl.
  • Representative compound of Formula I-p include without limitation SI 09, S122, and SI 23.
  • the compounds of Formula I, I-a', I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, 1-1, I-m, I-n, I-o, I-p, and Formula II can be used in methods that treat and/or prevent stress- induced cognitive dysfunction, and may also be used in compositions suitable for the treatment and/or prevention of stress-induced cognitive dysfunction.
  • the compounds used have structures as described by Formula I-a, I-b, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I- k-1, I-n, I-o, or I-p.
  • Another preferred embodiment relates to compounds of Formula I-a-1 :
  • n 0, 1, 2, 3, or 4;
  • Ri and R 2 together with the carbon and nitrogen to which they are respectively attached, form an unsubstituted or substituted heterocycle; or R 2 and R 3 together with the nitrogen and carbon to which they are respectively attached, form an unsubstituted or substituted heterocycle other than a piperazine; or
  • R 3 and R 4 together with the carbon atoms to which they are respectively attached, form an unsubstituted or substituted cycloalkyl or heterocyclic ring;
  • R4 is selected from the group consisting of R 5 and oxo;
  • each R 5 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, alkylaryl, and alkylheteroaryl;
  • Rg, R9, R 11 and Ri 2 are independently selected from the group consisting of R 5 , OR 5 , and -N(R 5 ) 2 ;
  • Ri 3 and Ri 4 are independently selected from the group consisting of R 5 , or Ri 3 and Ri 4 together with the N to which they are bonded may form an unsubstituted or substituted heterocycle;
  • Z is a halogen selected from F, CI, Br and I; and wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, alkylaryl, and alkylheteroaryl may be substituted or unsubstituted; wherein the nitrogen in the benzoxazepine ring may optionally be a quaternary nitrogen; and
  • the invention further provides a number of more preferred structures that fall within the general structure of formula I-a-1.
  • Preferred compounds of the present invention include:
  • each R 5 is independently hydrogen, or an unsubstituted or substituted alkyl, alkylaryl, aryl, or heterocyclyl.
  • R is methoxy
  • R 5 is hydrogen, or an unsubstituted or substituted alkyl, aryl, alkylaryl, heterocyclyl or heteroaryl; and wherein R and Ri4 are either each H or are bonded to make , wherein R d is CH 2 , NH, O,
  • Still other preferred compounds of the present invention include those of formula I-a-1, wherein
  • n 1 or 2
  • n 1
  • More preferred compounds of (a) include R 8 and R 9 being independently OR 5 . Also in (a)-(d), more preferred compounds of (a)-(d) include each R 5 being independently hydrogen, or an unsubstituted or substituted alkyl, alkylaryl, aryl, or heterocyclyl.
  • the more preferred compounds of the invention specifically include those of formula I-a-1, wherein:
  • n 1
  • R is OR 5 or OCZ 3 at position 7 of the benzoxazepine ring
  • Ri 3 and Ri 4 are either each H or are bonded to make ' , wherein Rd is CH 2 , NH,
  • Rj may optionally be a quaternary nitrogen; or R 2 and R 3 together with the nitrogen and carbon to which they are respectively attached, form an unsubstituted or substituted heterocycle other than a piperazine; or
  • N-benzo[l,3]dioxo-5-yl wherein the nitrogen in R d may optionally be a quaternary nitrogen; or R 2 and R 3 together with the nitrogen and carbon to which they are respectively attached, form an unsubstituted or substituted heterocycle other than a piperazine; or
  • N-benzo[l,3]dioxo-5-yl, or N-C( 0)OC(R 5 ) 3 , wherein the nitrogen in Rj may optionally be a quaternary nitrogen; or
  • n 1
  • R is OR 5 at position 6 of the benzoxazepine ring
  • R 2 and R 3 together with the nitrogen and carbon to which they are respectively attached, form an unsubstituted or substituted heterocycle other than a piperazine
  • R being OR 5 at position 7 of the benzoxazepine ring wherein each R 5 is independently hydrogen, or an unsubstituted or substituted alkyl, alkylaryl, aryl, or heterocyclyl.
  • Still other preferred compounds are those represented by the structure of any one or more of formula I-b- 1 , 1-c- 1 , 1-d- 1 , 1-e- 1 , 1-f- 1 , 1-g- 1 , 1-h- 1 , and I-i- 1 , and their
  • R, n and R 2 are as in formula I-a-1 and Rd is CH 2 , NH, O,
  • N-benzo[l,3]dioxo-5-yl, or N-C( 0)OC(R 5 ) 3 , wherein the nitrogen in Rj may optionally be a quaternary nitrogen.
  • the most preferred compounds of formula I-b-1 to I-i-1 include those where R is OR 5 at position 7 of the benzoxazepine ring wherein each R5 is independently hydrogen, or an unsubstituted or substituted alkyl, alkylaryl, aryl, or heterocyclyl.
  • R is methoxy at position 7 of the benzothiazepine ring.
  • Examples of compounds that may be used in conjunction with the invention include, without limitation, SI, S2, S3, S4, S5, S6, S7, S9, SI 1, S12, SI 3, S14, SI 9, S20, S22, S23, S24, S25, S26, S27, S36, S37, S38, S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S66, S67, S68, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, S100, S101,
  • each R is independently selected from the group consisting of H, halogen, -OH, -NH 2 , -NO 2 , -CN, -N3, -SO 3 H, acyl, alkyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-) aryl, (hetero-)arylthio, and (hetero-)arylamino may be substituted with one or more radicals independently selected from the group consisting of halogen, N,
  • each R is independently selected from the group consisting of H, halogen, -OH, -NH 2 , -N0 2 , -CN, -N 3 , -SO 3 H, acyl, alkyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-) aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl,
  • (hetero-)arylthio, and (hetero-) arylamino may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, -S-, -CN, -N 3 , -SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl.
  • R 5 is selected from the group consisting of -NR 16 , -(CH 2 ) z NRi 5 Ri6, NHNHRie, NHOH, -ORis, CONH 2 NHRi 6 , CONRi 6 , CH 2 X, acyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals
  • halogen independently selected from the group consisting of halogen, N, O, -S-, -CN, -N 3 , nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl.
  • the present invention provides use of the compounds of Formula II in the method of the invention.
  • Formula II is
  • Formula II is discussed also in US patent 7,718,644, the disclosure of which is incorporated herein in its entirety by reference.
  • the compounds of the invention reduce the open probability of RyR channels and decrease the calcium current through such channels by increasing binding of calstabin (FKBP12 or calstabin, and FKBP12.6 or calstabin2) binding affinity.
  • the compounds of the invention are useful for the treatment and/or prevention of disorders and conditions associated with abnormal function of RyR, particularly RyRl and RyR2 receptors, where such disorders and conditions are characterized by an increase in the open probability of, and in increase in the calcium current through, RyR channels.
  • a "decrease” or “disorder” in the level of RyR-bound FKBP in cells of a subject refers to a detectable decrease, diminution or reduction in the level of RyR-bound FKBP in cells of the subject.
  • Such a decrease is limited or prevented in cells of a subject when the decrease is in any way halted, hindered, impeded, obstructed or reduced by the administration of compounds of the invention, such that the level of RyR-bound FKBP in cells of the subject is higher than it would otherwise be in the absence of the administered compound.
  • the level of RyR-bound FKBP in a subject is detected by standard assays and techniques, including those readily determined from the known art (e.g. , immunological techniques, hybridization analysis, immunoprecipitation, Western-blot analysis, fluorescence imaging techniques and/or radiation detection, etc.), as well as any assays and detection methods disclosed herein.
  • the hybridization analysis occurs through the mechanism of protein-protein interaction.
  • the specific antibody which contains tag molecules can recognize the specific antigen, which is the target protein, and bind to the antigen.
  • the tagged molecules that are attached to the antibody can be detected using different techniques, e.g., fluorescence microscope.
  • a protein is isolated and purified from cells of a subject using standard methods known in the art, including, without limitation, extraction from the cells (e.g. , with a detergent that solubilizes the protein) where necessary, followed by affinity purification on a column, chromatography (e.g., FTLC and HPLC), immunoprecipitation (with an antibody), and precipitation (e.g., with isopropanol and a reagent such as Trizol). Isolation and purification of the protein is followed by electrophoresis (e.g., on an SDS- polyacrylamide gel).
  • a decrease in the level of RyR-bound FKBP in a subject, or the limiting or prevention thereof, is determined by comparing the amount of RyR-bound FKBP detected prior to the administration of JTV-519 or a compound of Formula I, I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1 , 1-1, 1-m, I-n, I-o, I-p, or Formula II, (in accordance with methods described below) with the amount detected a suitable time after administration of the compound.
  • a decrease in the level of RyR-bound FKBP in cells of a subject is limited or prevented, for example, by inhibiting dissociation of FKBP and RyR in cells of the subject; by increasing binding between FKBP and RyR in cells of the subject; or by stabilizing the RyR- FKBP complex in cells of a subject.
  • inhibiting dissociation includes blocking, decreasing, inhibiting, limiting or preventing the physical dissociation or separation of an FKBP subunit from an RyR molecule in cells of the subject, and blocking, decreasing, inhibiting, limiting or preventing the physical dissociation or separation of an RyR molecule from an FKBP subunit in cells of the subject.
  • the term “increasing binding” includes enhancing, increasing, or improving the ability of
  • phosphorylated RyR to associate physically with FKBP (e.g. , binding of approximately two fold or, approximately five fold, above the background binding of a negative control) in cells of the subject and enhancing, increasing or improving the ability of FKBP to associate physically with phosphorylated RyR (e.g., binding of approximately two fold, or,
  • a decrease in the level of RyR-bound FKBP in cells of a subject is limited or prevented by directly decreasing the level of phosphorylated RyR in cells of the subject or by indirectly decreasing the level of phosphorylated RyR in the cells (e.g., by targeting an enzyme (such as PKA) or another endogenous molecule that regulates or modulates the functions or levels of phosphorylated RyR in the cells).
  • the level of phosphorylated RyR in the cells is decreased by at least 10% in the method of the present invention. In another embodiment, the level of phosphorylated RyR is decreased by at least 20%.
  • the compounds of the present invention may be synthesized as described in published PCT application WO 07/024717 and U.S. patent application 11/506,285, the entire contents of which are hereby incorporated by reference.
  • the present invention provides use of compounds of the following formula for preparing many of the compounds disclosed herein:
  • Amyloid ⁇ -peptide (1-42) was prepared as described previously (Puzzo, D., et al, Amyloid-beta peptide inhibits activation of the nitric oxide/cGMP/cAMP -responsive element- binding protein pathway during hippocampal synaptic plasticity. J Neurosci, 2005. 25(29): p. 6887-97; Puzzo, D., et al, Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus. J Neurosci, 2008. 28(53): p. 14537-45.). This preparation is a mixed preparation containing monomers, trimers and tetramers.
  • Neuronal cells were isotonically lysed in 0.5 ml of a buffer containing 50 mM Tris- HC1 (pH 7.4), 150 mM NaCl, 20 mM NaF, 1.0 mM Na 3 V0 4 , and protease inhibitors.
  • DNP-hydrazone 2,4- dinitrophenylhydrazone
  • DNPH 2,4- dinitrophenylhydrazine
  • C57BL/6 mice, 3-4 months old, male are assigned into 4 groups: vehicle control, vehicle+S107, ⁇ control, and AP+S107.
  • SI 07 70-80mg/Kg/day is delivered through drinking water 3 weeks before behavioral tasks.
  • a cannula system (Plastics One Inc.,
  • the cannulas are fixed to the skull with acrylic dental cement.
  • the whole procedure is under anesthesia with 625-750 mg/kg Avertin LP.
  • mice receive a single bilateral injection of ⁇ 42 200nM or vehicle (aCSF) in a final volume of ⁇ over 1 min through the cannula 20 min before testing on day 1.
  • aCSF vehicle
  • mice receive injections 20 min before each section on both days. There is no injection on day 3. All mice are handled gently once a day for 3 days before the behavioral tasks.
  • Heterozygous transgenic 2576 mice expressing mutant human APP (K670N, M671L) are crossed with heterozygous PS1 mice that express mutant human PS1 (M146V; line 6.2). The offsprings, double-transgenic mice overexpressing APP/PS1, are compared with their wild-type littermates so that age and background strain are comparable. To identify the genotype of the animals, DNA extracted from tail tissue were used for PCR experiment.
  • the conditioning chamber (33 cm x 20 cm x 22 cm) is made of transparent Plexiglas on 2 sides and metal on the other 2. One of the metal sides has a speaker and the other has a 24- V light. It has a 36-bar insulated shock grid floor. The floor is removable and it is cleaned with 75% ethanol and then water after each test.
  • the conditioning chamber is located inside a sound-attenuating box (72cm x 51cm x 48cm).
  • a camera is placed on a tripod and connected to a computer monitoring system (Freezeframe software, MED Associates, Inc.) to record the experiment simultaneously through a clear Plexiglas window on the box. One animal at a time is present in the experimental room.
  • the radial-arm water maze consists of a round tank in 120 cm diameter with six swimming alleys (arms) 19 cm wide that radiate out from an open central area (40 cm in diameter).
  • a Plexiglas escape platform (10 x 10 cm) is located at the end of one of the arms, which is remained in the same location throughout the test.
  • the tank is filled with opaque water with non-toxic white water-color (Rich Art, NJ). Spatial cues are present on the walls and ceiling of the room.
  • Day 1 Training. There are 2 sections, with 15 trials per section and 60 sec for each trial. There is a 2-hour interval between sections. For each section, the escape platform is altered randomly in hidden and visible positions for 12 trials and it is kept in visible position for the last 3 trials. Errors are counted each time when mouse enters the wrong arm or needs more than 20 sec to reach the platform.
  • Day 2 Repeat the same tasks as on day 1, except the escape platform is hidden throughout the tests.
  • mice are sacrificed by cervical dislocation followed by decapitation. Hippocampi are quickly removed.
  • Transverse hippocampal slices 400 ⁇ are cut by a tissue chopper and transferred into an interface-type recording chamber where they are maintained at 29° C and perfused with artificial cerebrospinal fluid (aCSF) (124.0 mM NaCl, 4.4 mM KC1, 1.0 mM Na 2 HP0 4 , 25.0 mM NaHC0 3 , 2.0 mM CaCl 2 , 2.0 mM MgCl 2 , and 10 mM glucose, pH 7.4) continuously bubbled with 95% 0 2 and 5% C0 2 .
  • the field excitatory post-synaptic potentiations fEPSPs
  • fEPSPs are recorded from the CA1 region of the hippocampus by placement of both stimulating and recording electrodes in the CA1 stratum radiatum.
  • potentiation is induced using theta-burst stimulation (4 pulses at 100 Hz, with the bursts repeated at 5 Hz, and each tetanus including three 10-burst trains separated by 15 seconds). Responses are recorded for 2 hours after tetanization and measured as fEPSP slope expressed as percentage of baseline. V. Statistics
  • Figs. 1A-E show dose response to SI 07 treatment.
  • embodiment A are shown the cell survival rate (%) under different concentrations of compound SI 07 ( ⁇ (control), ⁇ , 10 ⁇ and ⁇ ) that is added to the 21 -day hippocampal primary culture cells. Cell number decreased after both 6 hours and 24 hours at 100 ⁇ of SI 07, so 1 ⁇ and 10 ⁇ was chosen for the experiments that generated the data for the embodiments B to E.
  • embodiment B are shown spine density (spine/micrometer) after the addition of SI 07 ( ⁇ , and 10 ⁇ ) to the hippocampal primary culture cells, with the density of spine count increasing after 1, 6 and 24 hours (*/? ⁇ 0.05).
  • embodiment C are shown cell survival rate after 24-hour addition of ⁇ ⁇ , 300nM, ⁇ ⁇ or 3 ⁇ to the hippocampal primary culture cells, wherein the higher concentrations caused a lower cell survival rate (*/? ⁇ 0.05).
  • embodiment D are shown spine density (spine/micrometer) after the addition of SI 07 ( ⁇ (control), ⁇ , and ⁇ ) to the hippocampal primary culture cells exposed to 300nM ⁇ , with the density increasing with greater concentrations of S 107 added (**/; ⁇ 0.05 ). Each panel shows the statistic result from 3 independent experiments.
  • embodiment E are shown spine diameter (micrometer) after the addition of SI 07 ( ⁇ (control), ⁇ , and 10 ⁇ ) to the hippocampal primary culture cells exposed to 300nM ⁇ , with the diameter increasing with greater concentrations of SI 07 added (**/; ⁇ 0.05 ).
  • Each panel shows the statistic result from 3 independent experiments.
  • Fig. 2 shows SI 07 protection against ⁇ .
  • Treatment with ⁇ at 300 nM in different time course increases expression of ROCK II, RhoA-GTP, p-CREB, and p-TAU, which was reduced by co-treatmwent with 10 ⁇ of SI 07.
  • a representative gel from 3 individual experiments using total ERK as loading control is shown.
  • Figs. 3A-C show that SI 07 improves memory and cognitive function in murine model of Alzheimer's tesed in the Radial Arm Water Maze (RAWM).
  • ⁇ infused mice treated with SI 07 ( ⁇ -8107) show a trend towards improvement in error reduction compared to ⁇ infused mice without SI 07 treatment ( ⁇ -Control).
  • Vehicle infused mice with SI 07 treatment (Veh-S107) and without SI 07 treatment (Veh-Control) groups were close to reaching the learning criterion (error ⁇ l) after block 10 on day 2.
  • Data presented are mean ⁇ S.E.M.
  • embodiments B and C are shown open pool task with visible platform.
  • Figs. 4 A and B show that SI 07 improves fear conditioning in Alzheimer's mouse model.
  • embodiment A are shown that there were no differences between groups at the baseline level of freezing behavior on day 1.
  • a 30 second tone a sound at 2,800 Hz and 85 dB
  • the percentage of freezing in ⁇ infusion group was lower than vehicle with and without S 107 treatment groups.
  • S 107 restored the freezing behavior to control level (* /? ⁇ 0.05).
  • embodiment B are shown that, during 2 minutes exposure to a "novel" (same chamber as in day-1 with decoration of plastic papers) chamber followed by a 3-minute cue (a sound at 2,800 Hz and 85dB), the ⁇ group showed lower percentage of freezing compared to controls on day 2. SI 07 rescued the freezing behavior significantly (* /? ⁇ 0.05). There were no differences between groups during 3 minutes cue period.
  • Figs. 5A and B show stress-induced PKA phosphorylation of RyRl and RyR2 in the brain and depletion of calstabin from the channel complexes.
  • Twelve 3 -month old WT male mice were randomized into 6 groups. Five groups were stressed as follows: Mice were subjected to chronic restraint stress (CRS) for 0 to 21 days in plexiglass restrainer tubes for 2 hrs each AM and PM.
  • CRS chronic restraint stress
  • RyR2 was immunoprecipitated from whole brain microsomes.
  • Figs. 6A-C show that mice subjected to chronic restraint stress for 3 weeks show a behavioral change in the number of open arm visits on the elevated plus maze.
  • Embodiment A are shown that the elevated plus maze has two open (o) and two closed (c) arms, and that at the start of the experiment the animal is placed in the center.
  • Open arm entries quantified as mean ⁇ SEM of time spent (embodiment B) and number of open arm visits for both the non- stressed control and the 3 -week restrained stressed group (n S each group) (embodiment C).
  • # P ⁇ 0.06 not significant trend, * P ⁇ 0.05.
  • Fig. 7 shows that SI 07 prevents calstabin2 depletion from PKA hyperphosphorylated
  • Calstabin2 binding to RyR2 which was significantly decreased in the untreated ISO group, was dose-dependently increased by SI 07 treatment.
  • Anti-RyR2 antibody was raised to a peptide corresponding to amino acids 1367-1380 of the mouse cardiac RyR2.
  • Figs. 8A-E show that SI 07 treatment corrects behavioral abnormalities in stressed mice.
  • A are shown that behavior was assessed by elevated plus maze open arm (o) versus closed arm (c) visits following 10 days of restraint stress for 3 hours per day each AM and PM (6 hours total/day).
  • B and C are shown open arm visits quantified as mean ⁇ SEM of time spent (embodiment B) and number of entry visits
  • Figs. 9A-C show fixing RyR leak prevents the stress induced depression of object recognition.
  • A are shown recognition trial consisting of a familiar (f) and a novel (n), unfamiliar object placed in an open field setup.
  • In embodiment B are shown exploration ratio prior to restrained stress was not significantly different between groups, and all groups performed significantly above 50% chance level.
  • embodiment C are shown exploration ratio after 10 days restraint stress was significantly decreased in the stress group (Stress), whereas in control (Control) and stress animals treated with SI 07 (Stress + SI 07), the ratio remained well above chance (right). These results suggest that SI 07 may ameliorate the stress-induced impairment of object recognition. Recognition with two identical objects (f) did not significantly differ between groups before and after restrained stress indicating similar object familiarization.
  • the exploration ratio represents the proportion of time that was spent exploring the novel versus familiar object [tnovel/(tnovel + tfamiliar)].
  • the dashed line represents the chance level of performance (i.e., a ratio of 0.50; no discrimination between sample and target object).
  • Figs. lOA-C show laser confocal imaging of a voltage-dependent Ca 2+ change in the mouse dentate gyrus of a brain slice (300 mm thick) loaded with the fluorescent Ca 2+ indicator Fluo-4 AM.
  • embodiment A is shown low magnification of the hippocampal dentate gyrus, acutely dissected using a standardized preparation method and kept under control conditions at 37°C in physiological saline solution (PSS) continuously gassed with O 2 /CO 2 to maintain pH and 02 tension.
  • PSS physiological saline solution
  • Fig. 11 shows single RyR2 R2474S/WT channels from knock -in mouse brains exhibit a gain-of- function defect that is rescued by SI 07 treatment.
  • RyR2 R2474S/WT which is
  • Figs. 12A-H show effect of ethanol on RyRl channels.
  • A is shown RyRl control channel activity.
  • B is shown channel activity during application of 4.4 mM ethanol.
  • C is shown channel activity in the presence of 180 microM H 2 O 2 .
  • H 2 O 2 was cumulatively added in cis chamber in the presence of ethanol.
  • D is shown channel activity in the presence of 180 microM H 2 O 2 and 8.8 mM ethanol.
  • E is shown RyRl control channel activity.
  • In embodiment F is shown channel activity during application of 180 microM H 2 O 2 .
  • G is shown channel activity in the presence of 180 microM H 2 O 2 and 8.8 mM ethanol.
  • RyRl channel activity was fully blocked by the application in cis of 10 microM ruthenium red.
  • Channel open probabilities are as indicated (Po) and the "c" indicates the closed state of the channel.
  • Channel openings are upwards. Representative of 3 or more channel preparations for each condition.
  • Fig. 13 shows that ethanol and H 2 O 2 synergistically activate leak in RyRl channels.
  • Ethanol (4.4 mM) alone in the cis (cytoplasmic) side of the channel did not increase the open probability (Po) of RyRl (second bar compared to first bar, control).
  • Po of RyRl was significantly increased (fourth and fifth bars). *, P ⁇ 0.05, **, P ⁇ 0.01.
  • Figs. 14A-D show "leaky” ryanodine receptors cause cognitive dysfunction.
  • learning curves of knock-in mice with leaky RyR2 channels (RyR2- S2808D), chronically stressed WT mice (Stress), control mice (Control), control mice treated with SI 07 (control + SI 07) and stressed mice treated with SI 07 (Stress + S 107) in a Morris water maze test.
  • Knock-in mice with leaky RyR2 channels (RyR2-S2808D) exhibit impaired learning to a degree similar to that observed in chronically stressed WT mice (Stress), as compared to control mice (Control), control mice treated with SI 07 (control + SI 07) and stressed mice treated with SI 07 (Stress + SI 07).
  • the stressed mice are subjected to 9-10 hours of overnight restraint stress daily for 3 weeks. Treatment with SI 07 which fixes the Ca 2+ leak in RyR2 channels improves learning in stressed mice.
  • embodiment B are shown bar graphs showing the total time spent in the target quardrant by knock-in mice with leaky RyR2 channels (RyR2-S2808D), chronically stressed WT mice (Stress), control mice
  • SI 07 Stress + SI 07
  • embodiment C are shown bar graphs showing the number of target crossings by knock-in mice with leaky RyR2 channels (RyR2-S2808D), chronically stressed WT mice (Stress), control mice (Control), control mice treated with SI 07 (control + SI 07) and stressed mice treated with SI 07 (Stress + SI 07).
  • embodiment D are shown learning curves of knock-in mice with leaky RyR2 channels (S2808D), WT mice (WT) and knock-in mice with leaky RyR2 channels treated with SI 07 (S2808D + SI 07) in a Morris water maze test. Treatment with SI 07 has no effect on learning in knock-in mice with leaky RyR2 channels. Figs.
  • 15A-C show that ryanodine receptor phosphrylation underlies cognitive dysfunction associated with ryanodine receptors.
  • learning curves of knock-in RyR2-S2808A mice in which the PKA phosphorylation site S2808 on RyR2 is inactivated by alanine substitution, without stress (S2808A) and with stress (S2808 + ST), as well as wildtype mice without stress (WT) and with stress (WT + ST).
  • Knock-in mice that cannot be phosphrylated by PKA exhibit active learning to a degree similar to that observed in WT mice without stress (WT), regardless whether they are stressed (S2808A + ST) or not (S2808A).
  • FIG. B are shown bar graphs showing the total time spent in the target quadrant for WT mice without stress (WT), WT mice with stress (WT + ST), knock-in S2808 A mice without stress (S2808 A), knock-in S2808 A mice with stress (S2808 A + ST), knock-in mice with leaky RyR2 channels (S2808D) and knock-in mice with leaky RyR2 channels treated with SI 07 (S2808D + SI 07).
  • FIG. C are shown bar graphs showing the number of entry to the target quadrant for wildtype mice without stress (WT), wildtype mice with stress (WT + ST), knock-in S2808A mice without stress (S2808A), knock- in S2808A mice with stress (S2808A + ST), knock-in mice with leaky RyR2 channels
  • Fig. 16 shows expression of RyRl and RyR2 in different regions of C57BL/6 adult male mouse brain.
  • expression of RyRl and RyR2 in hippocampus (HIP), cerebral cortex frontal pole (CCFP), lateral hypothalamic area (LHA), and cerebellum (CE) are shown magnified in a color-coded format to document differences in expression intensity revealed by in situ hybridization.
  • the expression intensity of RyR2 is maximal in the dentate gyrus and throughout CA1-CA3 of the hippocampal formation.
  • Other areas of maximal RyR2 expression include the CCFP and LHA, while the cerebellum granule cell layer (CE) signal indicates moderate expression.
  • CFP cerebellum granule cell layer
  • Figs. 17A and B show whole brain RyR2 biochemistry in wildtype and mutant mice with or without SI 07 treatment.
  • embodiment A are shown representative whole brain RyR2 immunoprecipitation and immunoblots showing amount of calstabin2 associated with RyR2, Cys-nitrosylation (Cys NO), RyR2 oxidation (DNP) and PKA phosphorylation level at S2808 (P2808) in wildtype mice (WT), wildtype mice under stress (WT, ST), wildtype mice under stress receiving SI 07 treatment (WT, ST + SI 07), mutant mice (S2808A) and mutant mice under stress (S2808A, ST).
  • embodiment B are shown representative whole brain RyR2 immunoprecipitation and immunoblots showing amount of calstabin2 associated with RyR2, Cys-nitrosylation (Cys NO) and RyR2 oxidation (DNP) in wildtype mice (WT) and mutant mice (S2808D).
  • SI 07 restored Calstabin 2 binding to RyR2 in wildtpe mice under stress.
  • Equivalent amounts of RyR2 were immunoprecipitated from SR whole brain microsomes using an anti-RyR2 antibody.
  • Figs. 18A and B show phosphorylation of CREB, ER and CaMKII in wildtype and mutant mice brain cells under stress or without stress.
  • A are shown representative whole brain immunoprecipitation and immunoblots showing phosphorylated and total amounts of CREB, ERK and CaMKII in wildtype (WT) and mutant mice (RyR2- S2808A) brain cells under stress or without stress.
  • B are shown bar graphs showing relative phosphorylation of CREB, ERK and CaMKII in wildtype and mutant mice brain cells under stress or without stress, determined by dividing the phosphorylated CREB, ERK or CaMKII signals by the total amount of the respective kinase that was
  • Figs. 19A-C show that SI 07 restores the neuronal long-term potentiation in stressed wildtype mice but not stressed knock-in mice with leaky RyR2 channels (S2808D).
  • the field excitatory post-synaptic potentiations (fEPSPs) are recorded from the CA1 region of the hippocampus by placement of both stimulating and recording electrodes in the CA1 stratum radiatum.
  • Long-term potentiation (LTP) is induced using theta-burst stimulation (4 pulses at 100 Hz, with the bursts repeated at 5 Hz, and each tetanus including three 10-burst trains separated by 15 seconds).
  • fEPSP slope expressed as percentage of baseline.
  • embodiment A long-term potentiation of neurons in WT mice without stress (Non-Stress), WT mice with stress (Stress), WT mice with stress receiving SI 07 treatment (Stress + SI 07). Stress reduces the field excitatory post-synaptic potentiations (fEPSPs), which is rescued by SI 07 treatment.
  • embodiment B are shown long-term potentiation of neurons in WT mice without stress (WT(C57BL/6)), knock-in S2808A mice (S2808A), and knock-in S2808A mice under stress (S2808A + ST).
  • fEPSPs field excitatory post-synaptic potentiations
  • S2808A mice Long-term potentiation of neurons in WT mice without stress (WT(C57BL)), knock-in mice with leaky RyR2 channels (S2808D) and knock-in mice with leaky RyR2 channels treated with SI 07 (S2808D + SI 07).
  • SI 07 treatment does not rescue the reduction of the field excitatory post-synaptic potentiations (fEPSPs) in stressed S2808D mice.
  • Figs. 20A-D show abnormal dentric spine morphology in knock-in mice with leaky RyR2 channels.
  • In embodiment D are shown expression of post synaptic density protein 95 (PSD) in mouse hippocampi of WT mice (WT) and knock-in mice with leaky RyR2 channels (S2808D). A representative gel from 3 individual experiments using total ER as loading control is shown.
  • PSD post synaptic density protein 95
  • Figs. 21 A and B show neuron calcium imaging in wildtype brain.
  • embodiment A are shown distribution of neurons and astrocytes in live hippocampal slice.
  • embodiment B are shown spontaneous calcium signals (Waves) from neurons of wildtype hippocampal CA1 area.
  • Figs. 22A and B show that abnormal ryanodine receptor activity is associated with neuronal degenerative disorders.
  • Animal models are generated by infusing vehicle or amyloid- ⁇ through cannula into hippocampal CA1 area in 3 months old male C57BL/6 mice.
  • embodiment A are shown representative whole brain RyR2 immunoprecipitation and immunoblots showing amount of calstabin2 associated with RyR2 and RyR2 oxidation (DNP) in controls (Ctrl, ages 67 and 88), Alzheimer's Disease patients (AD, both age 88) and Huntington's Disease patients (HD, ages 40 and 59).
  • DNP calstabin2 associated with RyR2 and RyR2 oxidation
  • AD Alzheimer's Disease patients
  • HD Huntington's Disease patients
  • the ⁇ infusion with SI 07 treatment group showed a trend towards improvement in error reduction compared to ⁇ infusion without SI 07 treatment group (Fig. 3 A).
  • Vehicle with and without SI 07 groups were close to reaching the learning criterion (error ⁇ l) after block 10 on day 2.
  • Data presented are mean ⁇ S.E.M.
  • Open pool task with visible platform was also measured. On day 3, visible platform training was performed to test visual and motor deficits in the same pool as in the 2-day water maze (Fig. 3B). However, there is no difference between between experimental groups, in terms of the time to reach platform.
  • SI 07 improves fear conditioning in Alzheimer's mouse model
  • mice with leaky RyR channels (RyRl-S2843D, RyR2-S2808D, calstabin2 deficient) and mice protected against stress induced leak (RyRl-S2843A) were used. Eight to ten weeks old male C57BL/6J mice were used and three groups of mice were tested for each experiment: Group 1 - controls, Group 2 - stress + tap water, Group 3 - stress + alcohol.
  • mice from the stress group were subjected to chronic restraint stress overnight for 3 weeks. Every Monday, Wednesday and Friday alcohol bottles were provided to these mice.
  • Alcohol consumption The effects of stress on alcohol consumption were tested.
  • Two- choice test procedure alcohol priming: Prior to the start of the experiment, mice were given 24 hr access to water and alcohol for 7 days using 2 feeding tubes (Dyets Inc, PA) inserted directly into the animal cage. Over these 7 days, the concentration of alcohol was gradually increased as follows: days 1-2: 3%; days 3-5: 6%; days 6-7: 10%. The position of water and alcohol tubes were changed on alternate days in order to avoid potential position bias.
  • Cognitive and behavioral function evaluation Morris water maze were used and elevated plus maze as well as fear conditioning and other cognitive and behavioral tests were used to test the effects of stress, vs. stress plus ethanol vs. stress plus ethanol plus SI 07 treatment (to prevent the RyR mediated intracellular Ca 2+ leak).
  • mice Stress-induced reinstatement of operant ethanol self-administration.
  • a separate group of mice were trained in the operant paradigm described above. Operant behavior was then extinguished during 6 weeks during which lever pressing did not result in the delivery of ethanol but activated the syringe pump.
  • mice were divided into two groups matched for responses during the final week of ethanol delivery and the final week of extinction. Half the animals were treated with SI 07 via osmotic pump, half vehicle via osmotic pump. Mice were placed in the chambers for a 30-min operant session using conditions identical to those used during extinction.
  • the Morris water maze is a test of spatial learning for rodents that relies on distal cues to navigate from start locations around the perimeter of an open swimming arena to locate a submerged escape platform. Spatial learning is assessed across repeated trials and reference memory is determined by preference for the platform area when the platform is absent. Performance in the Morris Water Maze is acutely sensitive to manipulations of the hippocampus. Subjects are placed in a circular pool of warm, opaque water in a random start location. An escape platform is hidden just under the surface of the water. During training trials, latency to find the platform location is recorded. During probe trials, the platform is removed, and the percentage of time spent in the quadrant that normally contains the platform is compared to the time spent in other quadrants. RESULTS
  • calstabin stabilizing subunit of the RyR channels that prevents Ca 2+ leak - calstabin is dissociated from the RyR channels by PKA phosphorylation of the channel
  • degree of stress required to dissociate calstabin from the RyR complex were also determined (Fig. 5), compared to non-stressed controls (0 Days).
  • 21 days of CRS demonstrated the lowest calstabinl (Fig. 5 A) and calstabin2 (Fig. 5B) binding to their respective RyRs compared to non-stress controls.
  • RyR-isoform specific antibodies rabbit polyclonal anti-RyRl raised to a peptide corresponding to amino acids 1327-1339 of mouse RyRl; rabbit polyclonal anti-RyR2 raised to a peptide
  • mice Sixteen 3 -month old C57BL/6J wild-type male mice were randomized to either the stress or control group. To minimize baseline anxiety or stress prior to restrained stress, mice were kept for at least one week under standard housing conditions and were habituated to the testing environment for another week. Using an elevated "plus" maze behavioral test of fear and anxiety, the animals were placed in the center of an elevated 4-arm maze in which 2 arms are open (o) and 2 are closed (c) (Fig. 6A). Using a video camera, the rodent's behavior was recorded and analyzed by a blinded observer. The number and duration of visits in each arms are measured.
  • SI 07 ⁇ -adrenergic agonist isoproterenol
  • SI 07 low dose 0.1 mg/ml
  • SI 07 high dose 0.25 mg/ml
  • the SI 07 medicated water was provided 4 days before the ISO treatment, and animals were sacrificed on day- 10.
  • RyR2 was immunoprecipitated from whole brain microsomes and assessed by immunoblot for PKA phosphorylation and calstabin2 binding.
  • the inventors have studied leaky RyR2 channels from a knock-in mouse that exhibits decreased seizure threshold, the RyR2-R2474S (RS) mice, a naturally occurring mutation in humans which causes arrhythmias and seizures.
  • RyR2RS/WT knock-in mice with norepinephrine (NOR 1 mg/kg LP.) and measured single-channel activity from brain vesicles in lipid bilayers.
  • the probability of RyR2 channels being open (Po) was significantly increased in NOR treated compared to control RyR2RS/WT mice.
  • Current amplitude histograms at 150 nM cis Ca 2+ revealed multiple subconductance open states consistent with a 'leaky' channel phenotype of the NOR group.
  • S107 treatment for 1 week of NOR treated RyR2RS/WT mice rescued the gain-of- function defect in the brain and confirmed SI 07 efficacy and specificity in vivo (Fig. 11).
  • knock-in S2808A mice in which the PKA phosphorylation site S2808 on RyR2 is inactivated by alanine substitution, were tested in
  • Fig. 15 A knock-in S2808A mice exhibit active learning to a degree similar to that observed in WT mice without stress, regardless whether they are under stress or not. Consistently, all three related kinases, CREB, ERK and CaMKII, show increased phosphylation in wildetype mice under stress (Fig. 18).
  • Ryanodine receptors are known to be expressed in the brain, especially in the hippocampus and cerebellum (Fig. 16).
  • calstabin2 associated with RyR2 is analyzed in whole brain samples, it was found that SI 07 restores calstabin 2 binding to RyR2 in wildtpe mice under stress (Fig. 17A).
  • SI 07 also restores the neuronal long-term potentiation in stressed wildtype mice but not stressed knock-in mice with leaky RyR2 channels (S2808D) (Figs. 19A-C).
  • Knock-in mice with leaky RyR2 channels have abnormal dentric spine morphology such as reduced spine density, as compared with wildtype controls.
  • the spine cells types and head and neck diameter in S2808D mice are the same as in wildtype mice (Figs. 20A-C).
  • the expression of post synaptic density protein 95 (PSD) in S2808D mice is reduced in comparison with that of the wildtype mice.
  • calstabin2 associated with RyR2 is
  • FIG. 22A Using the animal model for Alzheimer's Disease, it has been shown that SI 07 restores calstabin 2 binding to RyR2 in amyloid- ⁇ infused mice (Fig. 22B).

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Abstract

La présente invention concerne des procédés et des compositions utiles pour le traitement et/ou la prévention d'un disfonctionnement cognitif induit par le stress qui survient dans des situations telles qu'un abus de substances. Dans certains modes de réalisation, les procédés de la présente invention comprennent l'administration à un sujet d'une quantité thérapeutiquement ou prophylactiquement efficace d'un ou plusieurs composés de benzothiazépine, de benzoxazépine, de benzodiazépine ou de benzazépine définis par la formule générale (I).
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EA027922B1 (ru) * 2012-04-18 2017-09-29 Ле Лаборатуар Сервье Средства для лечения нарушений, вовлекающих модуляцию рецепторов рианодина
WO2013156505A1 (fr) 2012-04-18 2013-10-24 Les Laboratoires Servier Agents pour le traitement de troubles mettant en jeu la modulation de récepteurs de ryanodine
EP2708535A1 (fr) 2012-05-11 2014-03-19 Les Laboratoires Servier Agents pour le traitement de troubles impliquant la modulation de récepteurs de la ryanodine
WO2017203083A1 (fr) 2016-05-24 2017-11-30 Universidad Del País Vasco Triazoles pour la régulation de l'homéostase du calcium intracellulaire
US11377427B2 (en) 2016-05-24 2022-07-05 Universidad Del Pais Vasco Triazoles for regulating intracellular calcium homeostasis
US11504383B2 (en) 2021-01-08 2022-11-22 Armgo Pharma, Inc. Crystalline forms of a Ryanodine receptor modulator and uses thereof
US12076327B2 (en) 2021-01-08 2024-09-03 Armgo Pharma, Inc. Crystalline forms of a ryanodine receptor modulator and uses thereof
US12076326B2 (en) 2021-01-08 2024-09-03 Armgo Pharma, Inc. Crystalline forms of a ryanodine receptor modulator and uses thereof
US11717526B2 (en) 2021-05-20 2023-08-08 Armgo Pharma, Inc. Pharmaceutical compositions comprising a ryanodine receptor modulator and uses thereof

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