G9a / glp inhibitors and methods of use

Abstract

Described is small molecule N-(l-isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7- (3-(pyrrolidin-l-yl)propoxy)quinolin-4-amine)) (MS 1262) that inhibits methyltransferases G9a / GLP. This inhibitor can be used for the treatment of patients with G9a / GLP related diseases such as Alzheimer's Disease and Prader-Willi Syndrome.

Description

Attorney Docket No.: 27527-0222WO1 G9a / GLP INHIBITORS AND METHODS OF USE STATEMENT REGARDING GOVERNMENT FUNDING

[0001] This application claims priority to U.S. Provisional Application No.63 / 515,010, filed on July 21, 2023, the contents of which is hereby incorporated by reference in its entirety for all purposes.

[0002] This invention was made with government support under grants HD088626, AG071229 and GM133107 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] This disclosure is related to compounds that are small molecule inhibitors of G9a / GLP. The compounds disclosed are useful in treatment of various diseases including Alzheimer Disease (AD), Prader-Willi Syndrome (PWS) and cancers. More particularly, the disclosure is directed to (N-(1-isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3- (pyrrolidin-1-yl)propoxy)quinolin-4-amine)) (MS1262) and methods of use in the treatment of disease. BACKGROUND

[0004] Post-translation modifications (PTMs) of histones is a part of epigenetic regulation, which is critical for both the activation and repression of gene expression (Bhat et al., 2021). Three categories of proteins are involved in fulfilling these functions, the “writers”, which create the modifications; the “erasers”, which remove the modifications; and the “readers”, which recognize the modifications. Importantly, these proteins are implicated in a number of human diseases (Arrowsmith et al., 2012).

[0005] G9a (also known as EHMT2) and GLP (also known as EHMT1) are “writers” that catalyze the mono- and di-methylation of histone lysine 9 (H3K9). G9a and GLP are highly conserved in their catalytic SET domain (they share approximately 80% similarity in sequence).Attorney Docket No.: 27527-0222WO1 In the cell, G9a and GLP form a heterodimer to maintain the methyltransferase function. Dysregulation of G9a and GLP, especially G9a, are related to many human diseases. It has been reported that G9a overexpression is associated with cancer cell proliferation and metastasis in several types of cancer including but not limited to brain, breast, ovarian, lung, bladder, melanoma, and colorectal cancer (Kato et al., 2020). Moreover, it has been shown that G9a is involved in embryonic stem cell maintenance and T-cell differentiation, and is implicated in other diseases such as Alzheimer’s disease (AD) (Zheng et al., 2019), Sickle cell disease (SCD) and Prader-Willi syndrome (PWS) (Kim et al., 2017). Thus, small molecules targeting G9a / GLP would be invaluable for investigating the biological function of G9a / GLP, and for providing drug candidates for the treatment of disease.

[0006] To date, great progress has been made in developing small molecules targeting G9a / GLP. Those inhibitors include the first G9a / GLP chemical probe, UNC0638 (Vedadi et al., 2011), the first-in class G9a / GLP chemical probe suitable for in vivo study, UNC0642 (Liu et al., 2013), the first GLP selective inhibitor, MS012 (Xiong et al., 2017b), the first G9a / GLP inhibitor BIX01294 (Kubicek et al., 2007), UNC0224 (Liu et al., 2009), UNC0321 (Liu et al., 2010), BRD9539 (Yuan et al., 2012), E72 (Chang et al., 2010), A366 (Sweis et al., 2014), HKMTI-1-248 (Srimongkolpithak et al., 2014), CM-272 (Jose-Eneriz et al., 2017), EML741 (Milite et al., 2019), EPZ035544 (Campbell et al., 2017), compound 13 (Katayama et al., 2020), DS79932728 (Katayama et al., 2021) and RK-701(Takase et al., 2023). All these aforementioned inhibitors are reversible inhibitors which targets either the SAM binding site or the substrate binding pocket. In general, poor selectivity, high toxicity, short residence time and weak blood brain barrier penetration have been reported with these inhibitors. Therefore, there is a need for better inhibitors to be an alternative and effective therapeutic approach against G9a / GLP- mediated diseases. SUMMARY

[0007] Described herein is small molecule N-(1-isopropylpiperidin-4-yl)-6-methoxy-2- morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) (MS1262) that inhibits methyltransferases G9a / GLP. This inhibitor may be used for the treatment of patients with G9a / GLP related diseases such as Alzheimer’s Disease and Prader-Willi Syndrome.Attorney Docket No.: 27527-0222WO1

[0008] In one instance, a method of treating a neurodegenerative disease in a subject in need thereof, includes administering to a subject in need thereof a therapeutically effective amount of (N-(1-isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin- 4-amine)) (MS1262) or a pharmaceutically acceptable salt thereof. In one instance, the disease is Alzheimer's disease. In one instance, the disease is Prader-Willi Syndrome.

[0009] In one instance, a method of preventing Alzheimer's disease or Prader-Willi Syndrome in a subject in need thereof, includes administering to a subject in need thereof a therapeutically effective amount of (N-(1-isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7- (3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or a pharmaceutically acceptable salt thereof. In one instance, the (N-(1-isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1- yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition including a pharmaceutically acceptable carrier. In one instance, the (N-(1-isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1- yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof, is administered topically, rectally, nasally, buccally, vaginally, subdermally or ophthalmically. In one instance, the (N-(1-isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1- yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof, is administered in an orally acceptable dosage form, selected from capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In one instance, the (N-(1-isopropylpiperidin-4-yl)-6- methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof, is administered between one and six times per day. In one instance, the (N-(1-isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin- 4-amine)) or pharmaceutically acceptable salt thereof, is administered at a dose of between 0.001 and 1000 mg / kg / day.

[0010] In one instance, disclosed is (N-(1-isopropylpiperidin-4-yl)-6-methoxy-2-morpholino- 7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or a pharmaceutically acceptable salt thereof.

[0011] In one instance, a pharmaceutical composition, includes (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.Attorney Docket No.: 27527-0222WO1

[0012] In one instance, a pharmaceutical dosage form includes (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In one instance, the dosage form is selected from capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In one instance, the pharmaceutical dosage form includes (N-(1- isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4- amine)) in an amount of between 0.08 and 80,000 mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1. IC50 curves of MS1262 in G9a and GLP Thioglo biochemical assays.

[0014] Figure 2. Binding affinity of MS1262 in G9a and GLP ITC assays.

[0015] Figure 3. MS1262’s activity against other 21 epigenetic targets at 1 ?M.

[0016] Figure 4. Immunoblotting experiments of MS1262 in reducing H3K9me2 level in K562 cell line at indicated concentrations after 48 hours treatment.

[0017] Figure 5. PK results of MS1262.

[0018] Figure 6. Effect of MS1262 in activating the expression of PWS associated genes in fibroblasts derived from individuals with PWS.

[0019] Figure 7. Effect of MS1262 in activating PWS imprinting center in reporter mice model.

[0020] Figure 8. Effect of MS1262 in rescuing behavioral deficits in AD mice model.

[0021] Figure 9. Effect of MS1262 in increasing frequency but not amplitude of sEPSCs in DG granule cells of AD mice model without altering intrinsic excitability. DETAILED DESCRIPTION Synthesis and Testing of Compounds

[0022] The activity of novel synthesized compounds can be assessed using standard biochemical assays (G9a / GLP SAHH-coupled enzymatic assay), isothermal titration calorimetry (ITC) assays and Mt HotSpotTMradiometric assay. Cellular assays can then be used to assess theAttorney Docket No.: 27527-0222WO1 compound’s ability to reduce H3K9me2 levels, and reactivate PWS imprinting gene. Assays suitable for use in any or all of these steps are known in the art, and include, e.g., western blotting (WB), PCR, and q-PCR. Suitable cell lines for use in any or all of these steps are known in the art and include, K562, and PWS patient fibroblasts. Suitable mouse models for use in any or all of these steps are known in the art and include Snrpn-EGFP19 mice, the PWS mouse wild type (C57BL6) mice and FAD mice.

[0023] By way of non-limiting example, detailed synthesis protocols are described in the Examples for specific exemplary G9a / GLP inhibitors.

[0024] Pharmaceutically acceptable isotopic variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate isotopic variations of those reagents). Specifically, an isotopic variation is a compound in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature. Useful isotopes are known in the art and include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine. Exemplary isotopes thus include, e.g.,2H,3H,13C,14C,15N,17O,18O,32P,35S,18F, and36Cl.

[0025] Isotopic variations (e.g., isotopic variations containing2H) can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements. In addition, certain isotopic variations (particularly those containing a radioactive isotope) can be used in drug or substrate tissue distribution studies. The radioactive isotopes tritium (3H) and carbon-14 (14C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0026] Pharmaceutically acceptable solvates of the compounds disclosed herein are contemplated. A solvate can be generated, e.g., by substituting a solvent used to crystallize a compound disclosed herein with an isotopic variation (e.g., D2O in place of H2O, d6-acetone in place of acetone, or d6-DMSO in place of DMSO).

[0027] Pharmaceutically acceptable fluorinated variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagentsAttorney Docket No.: 27527-0222WO1 with appropriate fluorinated variations of those reagents). Specifically, a fluorinated variation is a compound in which at least one hydrogen atom is replaced by a fluoro atom. Fluorinated variations can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.

[0028] Pharmaceutically acceptable prodrugs of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (e.g., converting hydroxyl groups or carboxylic acid groups to ester groups). As used herein, a "prodrug" refers to a compound that can be converted via some chemical or physiological process (e.g., enzymatic processes and metabolic hydrolysis) to a therapeutic agent. Thus, the term "prodrug" also refers to a precursor of a biologically active compound that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject, i.e. an ester, but is converted in vivo to an active compound, for example, by hydrolysis to the free carboxylic acid or free hydroxyl. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in an organism. The term "prodrug" is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a subject. Prodrugs of an active compound may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound. Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of an alcohol or acetamide, formamide and benzamide derivatives of an amine functional group in the active compound and the like. Characterization of Exemplary G9a / GLP Inhibitors

[0029] A specific exemplary G9a / GLP inhibitor was characterized in biochemical assays, ITC assays, WB assays, PCR, q-PCR, pharmacokinetic (PK) studies and related disease animal models (Examples 2 – 10 Figs.1-9).

[0030] "Pharmaceutically acceptable salt" includes both acid and base addition salts. A pharmaceutically acceptable salt of any one of the bivalent compounds described herein isAttorney Docket No.: 27527-0222WO1 intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0031] "Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S.M. et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 66:1-19 (1997), which is hereby incorporated by reference in its entirety). Acid addition salts of basic compounds may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.

[0032] "Pharmaceutically acceptable base addition salt" refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium,Attorney Docket No.: 27527-0222WO1 magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N- methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. See Berge et al., supra. Pharmaceutical Compositions

[0033] In some aspects, the compositions and methods described herein include the manufacture and use of pharmaceutical compositions and medicaments that include one or more bivalent compounds as disclosed herein. Also included are the pharmaceutical compositions themselves.

[0034] In some aspects, the compositions disclosed herein can include other compounds, drugs, or agents used for the treatment of cancer. For example, in some instances, pharmaceutical compositions disclosed herein can be combined with one or more (e.g., one, two, three, four, five, or less than ten) compounds. Such additional compounds can include, e.g., conventional chemotherapeutic agents known in the art. When co-administered, the G9a / GLP inhibitor disclosed herein can operate in conjunction with conventional chemotherapeutic agents to produce mechanistically additive or synergistic therapeutic effects.

[0035] In some aspects, the pH of the compositions disclosed herein can be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the G9a / GLP inhibitors or its delivery form.

[0036] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier, adjuvant, or vehicle. As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. A pharmaceutically acceptable carrier,Attorney Docket No.: 27527-0222WO1 adjuvant, or vehicle is a composition that can be administered to a patient, together with a compound of the disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. Exemplary conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles include saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0037] In particular, pharmaceutically acceptable carriers, adjuvants, and vehicles that can be used in the pharmaceutical compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-?-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as ?-, ?-, and ?-cyclodextrin, may also be advantageously used to enhance delivery of compounds of the formulae described herein.

[0038] As used herein, the G9a / GLP inhibitors disclosed herein are defined to include pharmaceutically acceptable derivatives or prodrugs thereof. A “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, solvate, or prodrug, e.g., carbamate, ester, phosphate ester, salt of an ester, or other derivative of a compound or agent disclosed herein, which upon administration to a recipient is capable of providing (directly or indirectly) a compound described herein, or an active metabolite or residue thereof. Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds disclosed herein when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Preferred prodrugs include derivatives where a group that enhancesAttorney Docket No.: 27527-0222WO1 aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. Such derivatives are recognizable to those skilled in the art without undue experimentation. Nevertheless, reference is made to the teaching of Burger’s Medicinal Chemistry and Drug Discovery, 5thEdition, Vol.1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives.

[0039] The G9a / GLP inhibitor disclosed herein includes pure enantiomers, mixtures of enantiomers, pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, mixtures of diastereoisomeric racemates and the meso-form and pharmaceutically acceptable salts, solvent complexes, morphological forms, or deuterated derivatives thereof. The G9a / GLP inhibitor disclosed herein include, e.g., that derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate, trifluoromethylsulfonate, and undecanoate. Salts derived from appropriate bases include, e.g., alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)4+ salts. The disclosure also envisions the quaternization of any basic nitrogen-containing groups of the USP2 inhibitor disclosed herein. Water or oil-soluble or dispersible products can be obtained by such quaternization.

[0040] In some aspects, the pharmaceutical compositions disclosed herein can include an effective amount of one or more G9a / GLP inhibitors. The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of one or more compounds or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer). In some aspects, pharmaceutical compositions can further include one or more additional compounds, drugs, or agents used for the treatment of cancer (e.g., conventional chemotherapeutic agents) in amounts effective for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer).Attorney Docket No.: 27527-0222WO1

[0041] In some aspects, the pharmaceutical compositions disclosed herein can be formulated for sale in the United States, import into the United States, or export from the United States. Administration of Pharmaceutical Compositions

[0042] The pharmaceutical compositions disclosed herein can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA Data Standards Manual (DSM) (available at http: / / www.fda.gov / Drugs / DevelopmentApprovalProcess / FormsSubmissionRequirements / Electr onicSubmissions / DataStandardsManualmonographs). In particular, the pharmaceutical compositions can be formulated for and administered via oral, parenteral, or transdermal delivery. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0043] For example, the pharmaceutical compositions disclosed herein can be administered, e.g., topically, rectally, nasally (e.g., by inhalation spray or nebulizer), buccally, vaginally, subdermally (e.g., by injection or via an implanted reservoir), or ophthalmically.

[0044] For example, pharmaceutical compositions of this disclosure can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.

[0045] For example, the pharmaceutical compositions of this disclosure can be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this disclosure with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum toAttorney Docket No.: 27527-0222WO1 release the active components. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

[0046] For example, the pharmaceutical compositions of this disclosure can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, or other solubilizing or dispersing agents known in the art.

[0047] For example, the pharmaceutical compositions of this disclosure can be administered by injection (e.g., as a solution or powder). Such compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, e.g., as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, e.g., olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens, Spans, or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.

[0048] In some aspects, an effective dose of a pharmaceutical composition of this disclosure can include, but is not limited to, e.g., about 0.00001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,Attorney Docket No.: 27527-0222WO1 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000, or 10000 mg / kg / day, or according to the requirements of the particular pharmaceutical composition.

[0049] When the pharmaceutical compositions disclosed herein include a combination of a compound of the formulae described herein (e.g., a G9a / GLP inhibitors) and one or more additional compounds (e.g., one or more additional compounds, drugs, or agents used for the treatment of cancer or any other condition or disease, including conditions or diseases known to be associated with or caused by cancer), both the compound and the additional compound should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents can be administered separately, as part of a multiple dose regimen, from the compounds of this disclosure. Alternatively, those agents can be part of a single dosage form, mixed together with the compounds of this disclosure in a single composition.

[0050] In some aspects, the pharmaceutical compositions disclosed herein can be included in a container, pack, or dispenser together with instructions for administration. Methods of Treatment

[0051] The methods disclosed herein contemplate administration of an effective amount of a compound or composition to achieve the desired or stated effect. Typically, the compounds or compositions of the disclosure will be administered from about 1 to about 6 times per day or, alternately or in addition, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w / w). Alternatively, such preparations can contain from about 20% to about 80% active compound.

[0052] In some aspects, the present disclosure provides methods for using a composition comprising a G9a / GLP inhibitor, including pharmaceutical compositions (indicated below as ‘X’) disclosed herein in the following methods:

[0053] Substance X for use as a medicament in the treatment of one or more diseases or conditions disclosed herein (e.g., cancer, referred to in the following examples as ‘Y’). Use ofAttorney Docket No.: 27527-0222WO1 substance X for the manufacture of a medicament for the treatment of Y; and substance X for use in the treatment of Y.

[0054] In some aspects, the methods disclosed include the administration of a therapeutically effective amount of one or more of the compounds or compositions described herein to a subject (e.g., a mammalian subject, e.g., a human subject) who is in need of, or who has been determined to be in need of, such treatment. In some aspects, the methods disclosed include selecting a subject and administering to the subject an effective amount of one or more of the compounds or compositions described herein, and optionally repeating administration as required for the prevention or treatment of cancer.

[0055] In some aspects, subject selection can include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample for an indication that the subject is suitable for selection. In some aspects, the subject can be confirmed or identified, e.g. by a health care professional, as having had or having a condition or disease. In some aspects, suitable subjects include, for example, subjects who have or had a condition or disease but that resolved the disease or an aspect thereof, present reduced symptoms of disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), or that survive for extended periods of time with the condition or disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), e.g., in an asymptomatic state (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease). In some aspects, exhibition of a positive immune response towards a condition or disease can be made from patient records, family history, or detecting an indication of a positive immune response. In some aspects, multiple parties can be included in subject selection. For example, a first party can obtain a sample from a candidate subject and a second party can test the sample. In some aspects, subjects can be selected or referred by a medical practitioner (e.g., a general practitioner). In some aspects, subject selection can include obtaining a sample from a selected subject and storing the sample or using the in the methods disclosed herein. Samples can include, e.g., cells or populations of cells.

[0056] In some aspects, methods of treatment can include a single administration, multiple administrations, and repeating administration of one or more compounds disclosed herein as required for the prevention or treatment of the disease or condition from which the subject isAttorney Docket No.: 27527-0222WO1 suffering (e.g., a G9a / GLP -mediated disease). In some aspects, methods of treatment can include assessing a level of disease in the subject prior to treatment, during treatment, or after treatment. In some aspects, treatment can continue until a decrease in the level of disease in the subject is detected.

[0057] The term “subject,” as used herein, refers to any animal. In some instances, the subject is a mammal. In some instances, the term “subject,” as used herein, refers to a human (e.g., a man, a woman, or a child).

[0058] The terms “administer,” “administering,” or “administration,” as used herein, refer to implanting, ingesting, injecting, inhaling, or otherwise absorbing a compound or composition, regardless of form. For example, the methods disclosed herein include administration of an effective amount of a compound or composition to achieve the desired or stated effect.

[0059] The terms “treat”, “treating,” or “treatment,” as used herein, refer to partially or completely alleviating, inhibiting, ameliorating, or relieving the disease or condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder (e.g., cancer) are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disorder (e.g., cancer) refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with treatment by the compositions and methods of the present disclosure. In some embodiments, treatment can promote or result in, for example, a decrease in the number of tumor cells (e.g., in a subject) relative to the number of tumor cells prior to treatment; a decrease in the viability (e.g., the average / mean viability) of tumor cells (e.g., in a subject) relative to the viability of tumor cells prior to treatment; a decrease in the rate of growth of tumor cells; a decrease in the rate of local or distant tumor metastasis; or reductions in one or more symptoms associated with one or more tumors in a subject relative to the subject’s symptoms prior to treatment.

[0060] As used herein, the term “treating cancer” means causing a partial or complete decrease in the rate of growth of a tumor, and / or in the size of the tumor and / or in the rate of local or distant tumor metastasis, and / or the overall tumor burden in a subject, and / or any decrease in tumor survival, in the presence of an inhibitor (e.g., a G9a / GLP inhibitor) described herein.Attorney Docket No.: 27527-0222WO1

[0061] As used herein, the term “virus infection” means when an organism’s body is invaded by pathogenic viruses and infectious virus particle attach to and enter susceptible cells.

[0062] The terms “prevent,” “preventing,” “prevention,” “block”, and “blocking” as used herein, shall refer to a decrease in the occurrence of a disease or decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention may be complete, e.g., the total absence of disease or pathological cells in a subject. The prevention may also be partial, such that the occurrence of the disease or pathological cells in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the present disclosure. Exemplary G9a / GLP-mediated diseases that can be treated with G9a / GLP inhibitors include, for example, virus infection, AD, PWS, sickle cell disease, breast cancer, ovarian cancer, prostate cancer, colon cancer, pancreatic cancer, bladder cancer, liver cancer melanoma, and cervical cancer.

[0063] As used herein, the term “preventing a disease” (e.g., preventing cancer) in a subject means for example, to stop the development of one or more symptoms of a disease in a subject before they occur or are detectable, e.g., by the patient or the patient’s doctor. Preferably, the disease (e.g., cancer) does not develop at all, i.e., no symptoms of the disease are detectable. However, it can also result in delaying or slowing of the development of one or more symptoms of the disease. Alternatively, or in addition, it can result in the decreasing of the severity of one or more subsequently developed symptoms.

[0064] Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.

[0065] An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. Moreover, treatment of a subject with a therapeutically effective amount of the compounds or compositions described herein can include a single treatment or a series of treatments. For example, effective amounts can be administered at least once. The compositions can be administered from one or more times per day to one orAttorney Docket No.: 27527-0222WO1 more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.

[0066] Following administration, the subject can be evaluated to detect, assess, or determine their level of disease. In some instances, treatment can continue until a change (e.g., reduction) in the level of disease in the subject is detected. Upon improvement of a patient’s condition (e.g., a change (e.g., decrease) in the level of disease in the subject), a maintenance dose of a compound, or composition disclosed herein can be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced, e.g., as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

[0067] The present disclosure is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the disclosure or of any exemplified term. Likewise, the disclosure is not limited to any particular preferred embodiment or aspect described herein. Indeed, many modifications and variations may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the disclosure in spirit or in scope. The disclosure is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.

[0068] Example compounds are set forth in Table 1 below Table 1. Exampl Cpd ID Structure Namedi 3- - nolAttorney Docket No.: 27527-0222WO1 Chemistry General Procedures

[0069] All commercial chemical reagents and solvents were used for the reactions without further purification. Flash column chromatography was performed on Teledyne ISCO CombiFlash Rf+ instrument equipped with a 220 / 254 / 280 nm wavelength UV detector and a fraction collector. Normal phase column chromatography was conducted on silica gel columns with either hexane / ethyl acetate or dichloromethane / methanol as eluent. Reverse phase column chromatography was conducted on HP C18 RediSep Rf columns, and the gradient was set to 10% of acetonitrile in H2O containing 0.1% TFA progressing to 100% of acetonitrile. All final compounds were purified with preparative high-performance liquid chromatography (HPLC) on an Agilent Prep 1200 series with the UV detector set to 220 / 254 nm at a flow rate of 40 mL / min. Samples were injected onto a Phenomenex Luna 750 x 30 mm, 5 ?m C18 column, and the gradient was set to 10% of acetonitrile in H2O containing 0.1% TFA progressing to 100% of acetonitrile. All compounds assessed for biological activity have purity > 95% as determined by an Agilent 1200 series system with DAD detector and a 2.1 mm x 150 mm Zorbax 300SB-C185 ?m column for chromatography and high-resolution mass spectra (HRMS) that were acquired in positive ion mode using an Agilent G1969A API-TOF with an electrospray ionization (ESI) source. Samples (2 ?L) were injected onto a C18 column at room temperature, and the flow rate was set to 0.4 mL / min with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B. Nuclear magnetic resonance (NMR) spectra were acquired on Bruker DRX 400 MHz for proton (1H NMR) and 133 MHz for carbon (13C NMR). Chemical shifts for all compounds are reported in parts per million (ppm, ?). The format of chemical shift was reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant (J values in Hz), and integration. All final compounds had > 95% purity using the HPLC methods described above. EXAMPLES

[0070] Example 1 - Scheme 1. Synthesis of example 1Attorney Docket No.: 27527-0222WO1mg, according to previous reported procedures)(Liu et al., 2013), morpholine (17.4 ?L, 0.2 mmol), palladium acetate (4.5 mg, 0.02 mmol), BINAP (24.9 mg, 0.04 mmol)and tert-Butoxide Sodium (28.8 mg, 0.3 mmol) were mixed together in a microwave tube with THF (2 mL). The mixture was heated under microwave condition at 110°C for 30 min. After cooling to room temperature, the resulted mixture was diluted with dichloromethane and filtered. The filtration was collected and concentrated. The residue was purified by prep-HPLC to yield intermediate 2 as white solid (24 mg, 30% yield).1H NMR (600 MHz, Methanol-d4) ? 7.59 (s, 1H), 7.54 (s, 1H), 7.49 (s, 1H), 4.35 (t, J = 5.5 Hz, 2H), 4.03 (s, 3H), 3.92 – 3.88 (m, 4H), 3.88 – 3.82 (m, 6H), 3.50 (t, J = 7.2 Hz, 2H), 3.21 – 3.13 (m, 2H), 2.43 – 2.35 (m, 2H), 2.28 – 2.18 (m, 2H), 2.13 – 2.05 (m, 2H).

[0072] Intermediate 2 (24 mg, 0.06 mmol), 1-isopropylpiperidin-4-amine (30 ?L, 0.18 mmol),Pd2(dba)3 (11 mg, 12 ?mol), BINAP (15 mg, 24 ?mol) and Cs2CO3 (39 mg, 0.12 mmol)were mixed together in a microwave tube with dioxane (1 mL). The mixture was heated under microwave condition at 160°C for 30 min. After cooling to room temperature, the resulted mixture was diluted with dichloromethane and filtered. The filtration was collected and concentrated. The residue was purified by prep-HPLC to yield the titled compound as yellow solid (22 mg, 73% yield).1H NMR (600 MHz, Methanol-d4) ? 7.72 (s, 1H), 7.35 (s, 1H), 6.09 (s, 1H), 4.30 (t, J = 5.6 Hz, 2H), 4.23 – 4.12 (m, 1H), 4.00 (s, 3H), 3.88 (t, J = 4.9 Hz, 4H), 3.86 – 3.82 (m, , 2H), 3.71 (t, J = 4.9 Hz, 4H), 3.65 – 3.57 (m, 3H), 3.49 (t, J = 7.2 Hz, 2H), 3.34 – 3.31 (m, 2H), 3.20 – 3.12 (m, 2H), 2.45 – 2.32 (m, 4H), 2.25 – 2.20 (m, 2H), 2.18 – 2.04 (m, 4H), 1.42 (d, J = 6.6 Hz, 6H).13C NMR (151 MHz, Methanol-d4) ? 153.32, 153.21, 152.76, 147.84, 133.53, 107.96, 102.48, 100.27, 82.38, 66.50, 65.80, 58.23, 55.68, 54.03, 52.97, 47.70, 47.40, 46.50, 28.38, 25.22, 22.59, 15.55. HRMS (TOF): calcd for C29H46N5O3+[M + H]+512.3595, found 512.3621.

[0073] Table 2. Selected compounds’ potency in G9a biochemical assays Example No. IC50(nM)Attorney Docket No.: 27527-0222WO1 1 19 compounds’ potency in GLP biochemical assays.Example No. IC50 (nM) 1 6ed compounds’ binding affinity against G9a in ITC assays Example No. Kd(nM) 1 74ed compounds’ binding affinity against GLP in ITC assays Example No. Kd (nM) 1 19ure 1). IC50 curves of MS1262 in G9a and GLP Thioglo biochemical assays. MS1262’s IC50against G9a and GLP were determined by Thioglo assay with reported G9a / GLP inhibitor UNC0642 as control.

[0078] Example 3 (Figure 2). Binding affinity of MS1262 in G9a and GLP ITC assays.

[0079] Example 4 (Figure 3). MS1262’s activity against other 21 epigenetic targets at 1 ?M.

[0080] Example 5 (Figure 4). Immunoblotting experiments of MS1262 in reducing H3K9me2 level in K562 cell line at indicated concentrations after 48 hours treatment.

[0081] Example 6 (Figure 5). PK result of MS1262. Plasma and brain concentration of MS1262 over 4 h after i.p. injection at 5 mg / kg.

[0082] Example 7 (Figure 6). Effect of MS1262 in activating the expression of PWS associated genes in fibroblasts derived from individuals with PWS. (a) Schematic of in vitro treatment used in b, d, e. (b) Western blotting result of MS1262 and UNC0642 in reducing H3K9me2 level in PWS fibroblasts (left), and related quantification (right). (c) Schematic of genomic organization of PWS-associated imprinted domain at the human chromosome 15q11- q13 (IC, imprinting center). (d) PWS related mRNA level in PWS fibroblasts after treatment of MS1262 and UNC0642 at indicated concentrations.Attorney Docket No.: 27527-0222WO1

[0083] Example 8 (Figure 7). Effect of MS1262 in activating PWS imprinting center in reporter mice model. (a) Schematic of generation of the reported mice model and in vivo treatment. (b) Changes in weight gain in reporter mice with or without the treatment of indicated compounds. (c) Western blotting result of MS1262 and UNC0642 in reducing H3K9me2 level in the reported mice model (left), and related quantification (right). (d) PWS related mRNA level in reported mice after treatment of MS1262 or UNC0642

[0084] Example 9 (Figure 8). Effect of MS1262 in rescuing behavioral deficits in AD mice model. a. Experimental timeline for drug administration and NPR test. b. Depiction of the paradigm used to test memory and affective related behaviors. c. Locomotion in an open field was unaffected under wildtype, 5xFAD, vehicle, and chronic MS1262 treatment. d. Preference for the novel-located object during retrieval was significantly reduced in 5xFAD mice compared to wildtype controls and was completely rescued by chronic MS1262 treatment as measured by discrimination ratio (see methods for calculations). e. Time spent in the center of an open field was unaffected under wildtype, 5xFAD, vehicle, and chronic MS1262. f. Chronic MS1262 administration rescued anxiety-like behavior in 5xFAD mice back to wildtype levels demonstrated by increased time spent in the open arms of a zero maze. G. Depressive-like behavioral deficits in 5xFAD mice were rescued to wildtype levels after chronic MS1262 treatment as measured by immobile time during the forced swimming paradigm. Data are visualized as mean + / - standard error of the mean with each individual displayed as a point. Wildtype (n=7), 5xFAD (n=10), 5xFAD vehicle (n=14), 5xFAD MS1262 (n=12) mice were utilized for behavioral studies. Significance was assessed by ANOVA and Tukey’s posthoc test between each condition. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0085] Example 10 (Figure 9). Effect of MS1262 in increasing frequency but not amplitude of sEPSCs in DG granule cells of AD mice model without altering intrinsic excitability. a. Quantification of membrane capacity between 5XFAD mice treated with vehicle and MS1262 (n=21 / 22 cells for vehicle / MS1262; Two-tailed unpaired Student’s t test, p=0.216). b. Quantification of input resistance between 5XFAD mice treated with vehicle and MS1262 (n=21 / 22 cells for vehicle / MS1262; Two-tailed unpaired Student’s t test, P=0.109). c. Quantification of resting membrane potential between 5XFAD mice treated with vehicle and MS1262 (n=21 / 22 cells for vehicle / MS1262; Two-tailed unpaired Student’s t test, P=0.311). d. Mean (±SEM) number of action potential elicited in response to increasing step current fromAttorney Docket No.: 27527-0222WO1 dentate granule cells of 5XFAD mice injected with vehicle (black) or MS1262 (blue). (n=21 / 22 cells for vehicle / MS1262. Two-way ANOVA: main effect of Interaction, F12,533=0.123, P=0.999. Main effect of Group, F1,533=2.051, P=0.114). e. Representative traces of sEPSCs recorded from dentate granule cells derived from 5XFAD mice injected chronically with vehicle (left) or MS1262 (right). f-g. Cumulative probability (f) and average amplitude distribution (g) of sEPSCs (5xFAD-vehicle, n???16 cells from 3 mice; 5xFAD-MS1262, n???15 cells from 3 mice. P???0.2995, Mann Whitney test, two-sided). h-i. Cumulative probability distribution (h) and average frequency (i) of sEPSCs (5xFAD-vehicle, n???16 cells from 3 mice; 5xFAD-MS1262, ????15 cells from 3 mice. P???0.0171, Mann Whitney test, two-sided). *p???0.05, Data are visualized as mean???s.e.m. Biology G9a / GLP SAHH-coupled enzymatic assay

[0086] The assay was performed using the previously published protocol (Collazo et al., 2005). Briefly, tested compounds were pre-dissolved in assay buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 0.01% Triton X-100, 3 mM MgCl2, 0.1 mg / mL BSA) with 5 ?M of purified S- adenosylhomocysteine hydrolase (SAHH), 1 unit of adenosine deaminase (ADA) (Sigma- Aldrich, USA), 10 ?M of H3 peptide (1-25) (Anaspec, USA) and 5 nM of G9a (or GLP) as 30 ?L in a black 96-well plate. After 5 min, 20 ?L of SAM (25 ?M) (NEB, USA) in assay buffer was added. After incubation for 10 min at room temperature, thiol fluorescent probe IV (20 ?M, 50 ?L) (Sigma-Aldrich, USA) was added. After 10 min, the fluorescence of probe was measured at Ex400 / Em465 by Infinite M Plex (Tecan, USA). Expression and purification of G9a, GLP and SAHH

[0087] Catalytic domains of human G9a (913-1193) and GLP (982-1266) were cloned, expressed and purified according to the previously described protocol.(Xiong et al., 2017a) The full length of S. Solfataricus SAHH was cloned, expressed and purified using a published protocol.(Collazo et al., 2005) Isothermal titration calorimetry (ITC)Attorney Docket No.: 27527-0222WO1 Binding of selected compounds was analyzed using a MicroCal iTC200 (Malvern) in 50 mM Tris- HCl pH 7.5, 150 mM NaCl, 1% DMSO at 25 °C. After an initial 0.4 ?L injection, 13 injections (19 injections for MS8511N) from the syringe solution (500 ?M of compounds) were titrated into 300 ?L of the protein solution (50 ?M of G9a or GLP) in the cell, which was stirred at 750 rpm. The data were fitted by single binding site model using Microcal Origin 7.0 (Malvern). The reported values represent the mean ± SD from two independent measurements. Western blot analysis for H3K9me2

[0088] K562 cells were seeded in 6-well plates (Thermo Fisher Scientific, USA) at 5x105cells per well and treated with the indicated compound for 48 h. Cells were then lysed for 30 min on ice with RIPA buffer containing 1x proteasome / phosphatase inhibitor (Thermo Fisher Scientific, USA). Following lysis, samples were centrifuged for 10 min at 15000 rpm and 4?C. Supernatant was collected and mixed with 1x Laemmli buffer (Bio-Rad, USA) then heated at 100?C for 10 min. Protein concentration was quantified using the Pierce Rapid Gold BCA kit (Thermo Fisher Scientific, USA).10 ?g of each sample was loaded into a 4-20% Tris-Glycine gel (Bio-Rad, USA). SDS-PAGE was run at 90 V for 30 min then 120 V for 50 min. Following separation, proteins were transferred onto a PVDF membrane using Trans-Blot Turbo Transfer system (Bio-Rad, USA). Membranes were blocked using TBS Odyssey Blocking Buffer (LI- COR, USA) for 1 h at room temperature, then incubated in primary antibody (1:1000) overnight at 4 ?C. The next day, membranes were washed using 0.1% TBST and TBS then incubated with secondary antibody (1:10,000) for 1 h at room temperature. After washing, blots were imaged using Odyssey system (LI-COR, USA) and quantified using Image Studio (LI-COR, USA). Selectivity assays

[0089] Selectivity assays against other methyltransferases were performed by Reaction Biology Corp. (USA) using the same3H-labeled SAM based assay format as described above. All of experiments were performed using 1 µM of compound in duplicates. The compound enzyme inhibition effect was calculated as percentage of inhibition against control enzyme activity. Mouse pharmacokinetic studyAttorney Docket No.: 27527-0222WO1

[0090] For compounds administration: MS1262 (in its HCl salt form) was dissolved in a solution formulation of normal saline. Nine male C57BL / 6 mice (3 weeks old) were administered intraperitoneally with solution formulation of MS1262 at 5 mg / kg. MS152 (in its HCl salt form) was dissolved in a solution formulation of 5% v / v NMP, 5% v / v Solutol HS-15, and 90% v / v normal saline. Eighteen male C57BL / 6 mice were administered intraperitoneally with solution formulation of MS152 at 5 mg / kg or orally with solution formulation of MS152 at 50 mg / kg. MS93 (in its HCl salt form) was dissolved in a solution formulation of 5% v / v NMP, 5% v / v Solutol HS-15, and 90% v / v normal saline (for i.v.) and 10 % NMP, 5% Solutol HS-15, 50% PEG 400 and 35 % Captisol (10% W / V) (for p.o). Thirty-six male C57BL / 6 mice in total were split into two groups: Group 1 (n = 18) and Group 2 (n = 18). Group 1 were administered as an intravenous bolus injection through the tail vein with solution formulation of MS93 at 5 mg / kg and Group 2 were administered orally with a hazy solution formulation of MS152 at 25 mg / kg.

[0091] For samples collection and analysis: Blood samples (approximately 60 µL) were collected under light isoflurane anesthesia from a set of three mice at 0.5, 2 and 4 h (MS1262); 0.5, 2 and 8 hr (MS152); 0.083, 0.25, 0.75, 2, 8 and 24 h (MS93, i.v.); 0.52, 8, 12, 16 and 24h (MS93, p.o.). Plasma was harvested by centrifugation of blood and stored at -70 ? 10 °C until analysis, brain samples were collected from set of three mice at each time point the same as blood samples. Brain samples were homogenized using ice-cold phosphate buffer saline (pH-7.4) in a ratio of 2 (buffer):1(brain); and homogenates were stored below -70±10 ºC until analysis. Total homogenate volume was three times the brain weight. Plasma and brain samples were quantified by fit-for-purpose LC-MS / MS method. Pharmacokinetic analysis was performed using GraphPad Prism software in a way of nonlinear regression analysis. Compound concentrations in plasma at each time point are the average values from 3 test mice. Error bars represent ? SEM. PWS disease model experiments Animals

[0092] Animal handling for all experiments was conducted with an approved Institutional Animal Care and Use Committee (IACUC) protocol at Duke University. Snrpn-EGFP19 miceAttorney Docket No.: 27527-0222WO1 and the PWS mouse model with a paternal deletion from Snrpn to Ube3a (m+ / p?S-U)27 were previously described. Snrpn-EGFP and mice with a deletion from Snrpn to Ube3a (?S-U) mice were maintained on C57B6 / J and 129 / SvEv background, respectively. m+ / p?S-U mice and its littermates were produced by crossing wildtype females to heterozygous m ?S-U / p+ male mice. Cell culture

[0093] To generate primary mouse embryonic fibroblasts (MEFs) carrying maternal Snrpn- EGFP (mS-EGFP / p+), Snrpn-EGFP / + heterozygous females were crossed with wild-type males and embryos were isolated at E12.5 to E14.5 day. In addition, MEFs carrying paternal Snrpn- EGFP (m+ / pS-EGFP) were isolated from the embryos of wild-type females crossing with Snrpn- EGFP / + heterozygous males. Human PWS fibroblasts were obtained from Baylor College of Medicine cell repository and NIGMS Human Genetic Mutant Cell Repository. Mouse embryonic fibroblast cells were maintained in Dulbecco's modified Eagle's media (Gibco 11995-065) supplemented with 10% fetal bovine serum (Gibco 10082-147), 1% Gentamicin (Gibco 15710- 064), 1% Glutamine (Gibco 25030-149), 1% non-essential amino acid (Gibco 11140-050), 0.1% beta-mercaptoethanol (Gibco 21985-023), 100 Units / mL penicillin and 100 ?g / mL streptomycin (Gibco 15240-062) at 37oC and 5% CO2. Human fibroblast cells were maintained in Minimum Essential Medium Alpha media (Gibco 12571-063) supplemented with 10% fetal bovine serum (Gibco 10082-147), 1% L-Glutamine (Gibco 25030-081), 100 Units / mL penicillin and 100 micrograms / mL streptomycin (Gibco 15240-062) at 37oC and 5% CO2. In vitro and in vivo drug treatment

[0094] We cultured human fibroblast cells to ?80% confluence and treated them with compounds diluted in culture medium for 72 h. For treatment in the PWS model, we performed daily intraperitoneal (i.p.) injections to m+ / p???Ulitters starting at P7 and then for the following 5 d diluted in isotonic saline solution0.02% DMSO). We genotyped pups at the time of weaning or after their death. For testing chronic drug effects, we performed daily i.p. injection or oral administration to 6-week-old mS-EGFP / p+female mice for 7 consecutive days. RT–PCR and RT–qPCR

[0095] For reverse-transcription PCR (RT–PCR) and quantitative real-time RT–PCR (RT– qPCR), first we extracted total RNA from the fibroblasts and / or collected tissues (liver and brainAttorney Docket No.: 27527-0222WO1 from P14–P15 mice and 7, 10 and 18-week-old m+ / pS-Egfpmice) using Direct-zol RNA Miniprep kit (Zymo Research cat. no. R2070). We used 2 ?g of total RNA for single-strand cDNA synthesis with Superscript III reverse cat. no.18080-093) according to the manufacturer's protocols. The conditions for RT–PCR was 95 °C / 5 min, 35–40 cycles of 95 °C / 30 s, 56–60 °C / 60 s, 72 °C / 60 s. We performed quantification of target gene expression in a LightCycler480 instrument (Roche) using SsoAdvanced Universal SYBR green Supermix (Biorad cat. no.172-5271) according to the manufacturer's instructions. The primers that we used in this study are listed here.

[0096] SNRPN (forward, 5?-gctgcagcacattgactatagaat-3? (SEQ ID NO: 1); reverse, 5?- cacagtcatggataccaagttctc-3?), (SEQ ID NO: 2);

[0097] SNORD116 (forward, 5?-tggatcgatgatgagtcc-3? (SEQ ID NO: 3); reverse, 5?- tggacctcagttccgatgaga-3?), (SEQ ID NO: 4).

[0098] 116HG (forward, 5?-ctggtggatcccacaggt-3? (SEQ ID NO: 5); reverse, 5?- agaagcccacgccacata-3?), (SEQ ID NO: 6) .

[0099] 115HG (forward, 5?-cttcctcacaccctggtctc-3? (SEQ ID NO: 7);; reverse, 5?- gacttcaagaaatgcgtgctc-3?), (SEQ ID NO: 8).

[0100] NDN (forward, 5?-ggggtgggtcattatagtattcag-3? (SEQ ID NO: 9); reverse, 5?- acaaaaatccaagaaaggtagcac-3?), (SEQ ID NO: 10).

[0101] MAGEL2 (forward, 5?-ctaagaagctcatcaccgaag-3? (SEQ ID NO: 11); reverse, 5?- ggcagatacgaaaccaagttg-3?), (SEQ ID NO: 12).

[0102] ?-actin (forward, 5?-agagctacgagctgcctgac-3? (SEQ ID NO: 13); reverse, 5?- agcactgtgttggcgtacag-3?), (SEQ ID NO: 14).

[0103] mSnrpn (forward, 5?-ttggttctgaggagtgatttgc-3’ (SEQ ID NO: 15); reverse, 5?- ccttgaattccaccaccttg-3?), (SEQ ID NO: 16).

[0104] mSnord116 (forward, 5?-ggatctatgatgattcccag-3? (SEQ ID NO: 17); reverse, 5?- ggacctcagttccgatga-3?), (SEQ ID NO: 18).

[0105] m116HG (forward, 5?-ggttgcattccctttccagtatg-3? (SEQ ID NO: 19); reverse, 5?- cagcaattcccatgttccttacc-3?), (SEQ ID NO: 20).Attorney Docket No.: 27527-0222WO1

[0106] mUbe3a-ATS (forward, 5?-acagaacaataggtcaccaggtt-3? (SEQ ID NO: 21); reverse, 5?- aagcaagactgttcacctcat-3?), (SEQ ID NO: 22).

[0107] GFP (forward, 5?-acatgaagcagcacgacttct-3? (SEQ ID NO: 23); reverse, 5?- gacgttgtggctgttgtagttgta-3?) (SEQ ID NO: 24) and

[0108] GAPDH (forward, 5?-ggcaaattcaacggcacagt-3? (SEQ ID NO: 25); reverse, 5?- gggtctcgctcctggaagat-3?) (SEQ ID NO: 26). Chromatin immunoprecipitation assay

[0109] We analyzed histone methylations on the SNRPN locus in human fibroblasts by chromatin immunoprecipitation assay (ChIP) using the protocol as previously reported14,15,37. We performed ChIP assay using the ChIP-IT Express magnetic kit (Active Motif) according to the manufacturer's instructions, with modification for the fixation and reverse-cross-linking steps. Briefly, we prepared native chromatin without fixation and enzymatic digestions to average 150–500-bp-sized chromatin. We added 20 ?g of chromatin to the specific antibodies (2 ?g) or species control isotype antibodies for each immunoprecipitation reaction. We incubated the antibody-chromatin complexes with protein G magnetic beads for recovering chromatin immunoprecipitates. We purified RNase- and proteinase K–treated DNA using PCR purification columns (Promega). We quantified DNA recovery through real-time PCR performed on the LightCycler480 instrument (Roche) using SsoAdvanced Universal SYBR green Supermix (Biorad). We used the following antibodies: anti-rabbit acetylated H3 (Millipore 06-599), anti- mouse monoclonal histone H3 dimethyl K9 (Abcam 1220) and histone H3 trimethyl K9 (Millipore, 07-442), EHMT2 / G9a (Abcam ab40542). We performed qPCR reactions with the following cycling parameters: at 95 °C / 5 min followed by 40 cycles of 95 °C / 30 s, 60 °C / 60 s. We normalized data to the total input. The primers that we used in this study are listed here.

[0110] MAGEA2 (forward, 5?-gcctcaggatccccgtcccaat-3? (SEQ ID NO: 27); reverse, 5?- tggaaccggattctgcccggat-3?), (SEQ ID NO: 28).

[0111] CEN (forward, 5?-gtctctttcttgtttttaagctggg-3? (SEQ ID NO: 29); reverse, 5?- tgagctcattgagacatttgg-3?), (SEQ ID NO: 30).

[0112] NDN (forward, 5?-taaccctgttttccaggtatgg-3? (SEQ ID NO: 31); reverse, 5?- aagctgctgatgagaagaaacc-3?), (SEQ ID NO: 32).Attorney Docket No.: 27527-0222WO1

[0113] PWS-IC (forward, 5?-ctagaggccccctctcattgcaac-3? (SEQ ID NO: 33); reverse, 5?- cttcgcacacatccccgcctgagc-3?), (SEQ ID NO: 34).

[0114] SNORD116 (forward, 5?-tcttcaaatgtgcttggatcga-3? (SEQ ID NO: 35); reverse, 5?- tcttcaaatgtgcttggatcga-3?), (SEQ ID NO: 36).

[0115] U-SNRPN (forward, 5?-caatggaccaagagcattgata-3? (SEQ ID NO: 37); reverse, 5?- atagggtattgaaaccccgagt-3?), (SEQ ID NO: 38).

[0116] SNORD116dw (forward, 5?-tgagtcccacaaggaagttttt-3? (SEQ ID NO: 39); reverse, 5?- acattcaaagaggcaggacatt-3?), (SEQ ID NO: 40).

[0117] UBE3A (forward, 5?-ttgcttcctgagcaagtcataa-3? (SEQ ID NO: 41); reverse, 5?- tccgaaagcatgacatatcaac-3?), (SEQ ID NO: 42).

[0118] rhodopsin (forward, 5?- caagtcatgcagaagttagggg-3? (SEQ ID NO: 43); reverse, 5?- acccttataaagtgacctcccc-3?) (SEQ ID NO: 44); and

[0119] GAPDH (forward, 5?-gcatcacccggaggagaaaatcgg-3? (SEQ ID NO: 45); reverse, 5?- gtcacgtgtcgcagaggagc-3?) (SEQ ID NO: 46). AD disease model experiments

[0120] Human cerebral organoid cultures. A familial AD patient-derived iPSC line with the APP V717I mutation (F033K; male) and a sex- and age-matched healthy control (C-03; male) iPSC line (provided by Dr. Chadwick Hales’ laboratory at Emory University) were cultured on irradiated MEFs in human iPSC medium composed of D-MEM / F12 (Invitrogen), 20% Knockout Serum Replacement (KSR, Invitrogen), 1X Glutamax (Invitrogen), 1X MEM Non-essential Amino Acids (NEAA, Invitrogen), 100 µM ?- Mercaptoenthanol (Invitrogen), and 10 ng / ml human basic FGF (bFGF, PeproTech) as described (Kang et al., 2021; Kuehner et al., 2021; Wen et al., 2014). Forebrain-specific organoids were generated as described (Kang et al., 2021; Kuehner et al., 2021; Qian et al., 2016). Briefly, human iPSC colonies were detached from the feeder layer with 1 mg / ml collagenase treatment for 1 hour and suspended in embryonic body medium composed of FGF-2-free iPSC medium supplemented with 2 µM Dorsomorphin and 2 µM A-83 in nontreated polystyrene plates for 4 days with a daily medium change. On days 5-6, half of the medium was replaced with induction medium consisting of DMEM / F12, 1X N2 Supplement (Invitrogen), 10 ?g / ml Heparin (Sigma), 1X Penicillin / Streptomycin, 1X Non-Attorney Docket No.: 27527-0222WO1 essential Amino Acids, 1X Glutamax, 4 ng / ml WNT-3A (R&D Systems), 1 ?M CHIR99021 (Tocris), and 1 ?M SB-431542 (Tocris). On day 7, organoids were embedded in Matrigel (BD Biosciences) and continued to grow in induction medium for 6 more days. On day 14, embedded organoids were mechanically dissociated from Matrigel by pipetting up and down onto the plate with a 5 ml pipette tip. Typically, 10-20 organoids were transferred to each well of a 12-well spinning bioreactor (Spin?) containing differentiation medium, consisting of DMEM / F12, 1X N2 and B27 Supplements (Invitrogen), 1X Penicillin / Streptomycin, 100 µM ?- Mercaptoenthanol (Invitrogen), 1X MEM NEAA, 2.5 ?g / ml Insulin (Sigma). At day 71, differentiation medium was exchanged with maturation medium, consisting of Neurobasal (Gibco), 1X B27 Supplement, 1X Penicillin / Streptomycin, 1X ?-Mercaptoenthanol, 0.2 mM Ascorbic Acid, 20 ng / ml BDNF (Peprotech), 20 ng / ml GDNF (Peprotech), 1 ng / ml TFG? (Peprotech), and 0.5 mM cAMP (Sigma). All media were changed every other day.

[0121] Experimental animals. All animal procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill. 5xFAD (C57BL / 6J) and wild-type (C57BL / 6J) littermate controls (16-24-weeks-old) were obtained from the Jackson laboratory. All 5xFAD mice were heterozygous. Both male and female mice were used and sex was matched in the various groups. No immune deficiencies or other health problems were observed in these lines, and all animals were experimentally and drug-naïve before use. Animals were group housed and bred in a dedicated husbandry facility with 12 / 12 hour light-dark cycles with ad libitum food and water. All mice were under veterinary supervision. Behavioral experiments were performed in the light phase. Once drug or vehicle administration began, animals were moved to a satellite housing facility with the same light-dark cycle.

[0122] Drug treatment. The G9a inhibitor MS1262 was dissolved in DMSO to 10 mg / mL and aliquoted into single doses and stored at -20°C. Immediately prior to injection, these aliquots were thawed and diluted in 0.9% saline. The final solution that was injected intraperitoneally consisted of 1% DMSO and equated to a 1 mg MS1262 / kg of animal weight. Control animals received 1% DMSO in 0.9% saline at weight matched volumes. Mice were randomly selected for MS1262 or vehicle treatment and received 1 injection every 3.5 days for 6 weeks.Attorney Docket No.: 27527-0222WO1 Behavioral tests

[0123] Handling. Mice were handled five days a week for 6 weeks for 5 min / day leading up to behavioral testing.

[0124] Open Field and Habituation. On days 1 and 2, mice were placed in an empty open field environment (45 cm square plastic chamber) for 10 minutes. After each test, the chamber was cleaned with 70% ethanol to eliminate scents from previously tested mice. The first 5 minutes of the day 1 test was analyzed by Noldus Ethovision XT to monitor animal position and locomotion. A 25 cm square was used to indicate the center of the chamber when analyzing the recordings. Both total locomotion and time spent in the center of the open field were quantified by the Noldus Ethovision XT program.

[0125] Novel place recognition (NPR). On day 3 of behavioral testing, also known as the encoding phase, two identical glass cylinders (height 4 cm, base diameter 1.5 cm) were fixed to the chamber floor (to prevent object movement) on the same side of the chamber 20 cm apart from each other. Animals were allowed to freely explore the chamber for 5 minutes and could interact with the objects while being recorded. Most animals showed no preference for a single object and the locations of the objects were randomized across mice. On day 4 of behavioral testing, also known as the retrieval phase, the position of one of the objects was moved to the opposite side of the chamber, and animals were recorded for 5 minutes while freely exploring the chamber and interacting with the objects. Videos were scored manually using a separately trained researcher who was blinded to the treatment group. Time spent with each object in both encoding and retrieval were scored. Any mouse that showed a preference for one object over the other (defined by interacting with one object for more than double the time of interacting with the other) during the encoding phase was not included in the analysis. Furthermore, mice that did not spend at least 2 seconds of total object interaction time (IT) were not included in the analysis. The discrimination ratio (DR) was calculated in the following way: DR = (ITnovel location – ITfamiliar location) / (ITnovel location + ITfamiliar location).

[0126] A DR of 0 indicates no preference for either object, and a DR of 0.33 indicates spending twice as much time with the object in the novel location compared with the object in the familiar location.Attorney Docket No.: 27527-0222WO1

[0127] Elevated zero maze. The apparatus was an elevated white plastic ring platform (width of ring 6 cm, outer diameter 60 cm). The entire ring was elevated 60 cm off the ground. Each animal was placed in the closed arm to start the trial and was recorded for 5 minutes. Time spent in the open arm sections was scored by a trained, blinded researcher. After each trial, the apparatus was cleaned with 70% ethanol.

[0128] Forced Swim. The apparatus was an acrylic cylinder (diameter 20 cm, height 30 cm) filled with room temperature (23?1oC) water to a depth of 20 cm. Each mouse was recorded during a 5-minute swimming trial, and the video was later scored manually by a trained, blinded researcher for time spent immobile. Time spent immobile was defined as when all four paws of the mouse remained immobile. After each trial, the apparatus was filled with fresh water.

[0129] Microdissection of hippocampi. After 6 weeks of intermittent MS1262 or vehicle treatment, animals were anesthetized using a 5% isoflurane in oxygen mixture until the animal was no longer responsive to a toe pinch. Animals were then transcardially perfused with ice-cold PBS and the brain was isolated. The brain was sliced bilaterally across the sagittal midline. Each half was then taken, and the hippocampus was carefully dissected. Both hippocampi were placed in a cryogenic tube and flash frozen in liquid nitrogen and placed at -80oC.

[0130] Slice preparation. Acute slices were prepared from 5.25 month-5.5 month male 5xFAD mice treated with MS1262 (1 mg / kg) or vehicle as described previously. Animals were anesthetized with isoflurane (5% in O2) and transcardially perfused with ice-cold oxygenated artificial cerebrospinal fluid (ACSF) containing the following items (in mM): 92 NMDG, 30 NaHCO3, 25 glucose, 20 HEPES, 10 MgSO4, 5 sodium ascorbate, 3 sodium pyruvate, 2.5 KCl, 2 thiourea, 1.25 NaH2PO4, and 0.5 CaCl2(pH 7.3, 310 mOsm). Brains were rapidly removed, and acute transverse hippocampal slices (280??m) were cut using a Leica vibratome (VT1200, Germany). Next, slices were warmed to 34.5°C for 8 minutes. Then, slices were maintained in the holding chamber containing HEPES ACSF (in mM): 92 NaCl, 30 NaHCO3, 25 glucose, 20 HEPES, 5 sodium ascorbate, 3 sodium pyruvate, 2.5 KCl, 2 thiourea, 2 MgSO4, 2 CaCl2, 1.25 NaH2PO4 (pH 7.3, 10 mOsm) at ambient temperature for at least 1 hour before recording. Electrophysiological recordings were made at 32°C using a heater controller (TC-324C, Warner Instruments) in ACSF containing (in mM): 125 NaCl, 26 NaHCO3, 20 glucose, 2.5 KCl, 2 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4, (pH 7.3, 310 mOsm). The flow rate was 2 ml / min.Attorney Docket No.: 27527-0222WO1

[0131] Electrophysiology. Slices were visualized on a fixed-stage upright microscope (Olympus BX51WI) equipped with ×4 and ×40 objectives and differential interference contrast optics, infrared illumination, and an infrared-sensitive camera. Whole-cell patch-clamp recordings were performed with glass pipettes with resistance of 4.0–6.0 M? when filled with internal solution containing (in mM): 130 K-gluconate, 20 HEPES, 4 MgCl2, 4 Na-ATP, 2 NaCl, 0.5 EGTA, 0.4 Na-GTP (pH 7.2, 290 mOsm). Series resistance (Rs) was monitored throughout all experiments, and cells with Rs changes over 20% were discarded. All recordings were made from granule cells located in the middle or outer layer of the dentate gyrus. For spontaneous excitatory postsynaptic currents (sEPSCs) recordings, dentate granule cells were held at -70 mV in voltage-clamp mode and bicuculine (20??M; GABAA receptor antagonist; Tocris Bioscience) was added to the ACSF. Intrinsic cellular properties were recorded in current-clamp mode. To test neuronal input resistance, hyperpolarizing current pulses (20 pA, 200 ms) were applied to the neurons. Resting membrane potential was measured as the membrane potential baseline value obtained in current-clamp mode in the absence of current injection. The current-voltage relationship experiments (to evaluate action potential firing rate) consisted of a series of current injections (500-ms duration) between 0 pA and +240 pA in 20 pA delivered in step-wise increments. Data were acquired with a Multiclamp 700B amplifier and digitized with a Digidata 1440A using pCLAMP 10.7 acquisition software (Molecular Devices).Attorney Docket No.: 27527-0222WO1 References: Arrowsmith, C.H., Bountra, C., Fish, P.V., Lee, K., and Schapira, M. (2012). Epigenetic protein families: a new frontier for drug discovery. Nat Rev Drug Discov 11, 384-400.10.1038 / nrd3674. Bhat, K.P., Kaniskan, H.U., Jin, J., and Gozani, O. (2021). 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Claims

Attorney Docket No.: 27527-0222WO1 CLAIMS 1. A method of treating a neurodegenerative disease in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of (N-(1- isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4- amine)) (MS1262) or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the neurodegenerative disease is Alzheimer's disease.

3. A method of preventing Alzheimer's disease in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of (N-(1- isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4- amine)) or a pharmaceutically acceptable salt thereof.

4. The method of any one of claims 1 - 3, wherein the (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition including a pharmaceutically acceptable carrier.

5. The method of any one of claims 1 - 4, wherein the (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof, is administered topically, rectally, nasally, buccally, vaginally, subdermally or ophthalmically.

6. The method of any one of claims 1 - 5, wherein the (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof, is administered in an orally acceptable dosage form, selected from capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.Attorney Docket No.: 27527-0222WO1 7. The method of any one of claims 1 - 6, wherein the (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof, is administered between one and six times per day.

8. The method of any one of claims 1 - 7, wherein the (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof, is administered at a dose of between 0.001 and 1000 mg / kg / day.

9. The method of claim 1, wherein the neurodegenerative disease is Prader-Willi Syndrome.

10. A method of preventing Prader-Willi Syndrome in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of (N-(1- isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4- amine)) or a pharmaceutically acceptable salt thereof.

11. The method of either of claims 9 – 10, wherein the (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof is administered as a pharmaceutical composition including a pharmaceutically acceptable carrier.

12. The method of any one of claims 9 - 11, wherein the (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof, is administered topically, rectally, nasally, buccally, vaginally, subdermally or ophthalmically.

13. The method of any one of claims 9 - 12, wherein the (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof, is administered in an orally acceptable dosage form, selected from capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.Attorney Docket No.: 27527-0222WO1 14. The method of any one of claims 9 - 13, wherein the (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof, is administered between one and six times per day.

15. The method of any one of claims 9 - 14, wherein the (N-(1-isopropylpiperidin-4- yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or pharmaceutically acceptable salt thereof, is administered at a dose of between 0.001 and 1000 mg / kg / day.

16. (N-(1-isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1- yl)propoxy)quinolin-4-amine)) or a pharmaceutically acceptable salt thereof.

17. A pharmaceutical composition, comprising (N-(1-isopropylpiperidin-4-yl)-6- methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

18. A pharmaceutical dosage form, comprising (N-(1-isopropylpiperidin-4-yl)-6- methoxy-2-morpholino-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-amine)) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

19. The pharmaceutical dosage form of claim 18, wherein the dosage form is selected from capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.

20. The pharmaceutical dosage form of claim 18, wherein the dosage form comprises a dosage of (N-(1-isopropylpiperidin-4-yl)-6-methoxy-2-morpholino-7-(3-(pyrrolidin-1- yl)propoxy)quinolin-4-amine)) in an amount of between 0.08 and 80,000 mg.

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