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WO2025215108A1 - Inhibiteurs doubles de g9a/gsk-3ss - Google Patents

Inhibiteurs doubles de g9a/gsk-3ss

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Publication number
WO2025215108A1
WO2025215108A1 PCT/EP2025/059782 EP2025059782W WO2025215108A1 WO 2025215108 A1 WO2025215108 A1 WO 2025215108A1 EP 2025059782 W EP2025059782 W EP 2025059782W WO 2025215108 A1 WO2025215108 A1 WO 2025215108A1
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WO
WIPO (PCT)
Prior art keywords
methyl
tetrahydropyrimidine
carboxylate
ethyl
oxo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/059782
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English (en)
Inventor
Cristian Gaspar GRIÑAN FERRE
Aina BELLVER SANCHIS
Mercè PALLÀS LLIBERIA
Marta RIBALTA VILELLA
Santiago VÁZQUEZ CRUZ
María Carmen ESCOLANO MIRÓN
Ainoa SANCHEZ ARFELIS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitat de Barcelona UB
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Universitat de Barcelona UB
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Application filed by Universitat de Barcelona UB filed Critical Universitat de Barcelona UB
Publication of WO2025215108A1 publication Critical patent/WO2025215108A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention refers to compounds which are potent and specific G9a/GSK3p inhibitors, as well as to pharmaceutical compositions comprising these compounds and uses thereof in the treatment or prevention of diseases or conditions related to G9a/GSK3p overexpression.
  • AD Alzheimer’s disease
  • MTDLs Multiple Targeting Directed Ligands
  • methyltransferase G9a and glycogen synthase kinase serve as drivers of cognitive impairment, leading to synaptic plasticity reduction, autophagy dysfunction, increasing Tau pathology, OS, and neuroinflammation collectively contributing to and/or resulting in age-related cognitive decline and neurodegenerative disease, particularly AD.
  • G9a and its repressive mark increase with age and produce memory impairment.
  • H3k9me2 is related to the repression of genes implicated in synaptic plasticity, learning, and memory formation (A. Bellver-Sanchis A, et al., “G9a Inhibition Promotes Neuroprotection through GMFB Regulation in Alzheimer's Disease”, Aging Dis. 2024, 15, 311-337).
  • G9a and its repressive mark (H3k9me2) induce autophagic dysfunction and oxidative stress.
  • known G9a inhibitors tend to produce high toxicity, low efficacy in vivo and in vitro studies, and poor brain-blood barrier (BBB) permeability (A.
  • GSK-3P increases with age, is overexpressed in AD patients, modulates tau hyperphosphorylation, induces AB toxicity and neuroinflammation, and promotes loss of memory consolidation, neurogenesis, and synaptic plasticity (E. Lauretti, et al., “Glycogen synthase kinase-3 signaling in Alzheimer's disease”, Biochim. Biophys. Acta Mol. Cell Res. 2020, 1867, 118664). GSK-3p inhibitors are usually associated with safety problems and have shown limited efficacy in clinical trials (S. M.
  • the present invention provides dual G9a/GSK-3p inhibitors capable of modulating simultaneously epigenetic alterations and tau pathology, two crucial causes of AD.
  • Such dual inhibitors can reduce adverse effects and safety/toxicity problems, among other things. Therefore, our dual approach is an innovative and promising multifaceted therapeutic strategy for AD treatment and other G9a/GSK-3p-mediated disorders.
  • the present invention in a first aspect, refers to a compound of formula (I), or a salt, solvate or stereoisomer thereof: wherein: R is one or more radicals independently selected from the group consisting of: H; OH; CN; fluorine; chlorine; (Ci-Cw)haloalkyl; -0-(Ci-Cio)alkyl; (Ci-Cio)alkyl; -O-(Ci- Cw)haloalkyl; SF5; S(O)2R’I; NR ⁇ R’s; CONR ⁇ R’s; and COOR’e;
  • R'i, R'2, R’3, R’4, R’s and R’e are independently selected from the group consisting of: -H, (Ci-Cw)alkyl; (Ci-Cw)haloalkyl; and (C3-C8)cycloalkyl;
  • X represents O or S
  • Z represents O or NH
  • R 1 is selected from the group consisting of: a (Ci-Cw)alkyl; or a 4-halophenyl ring; for use in preventing or treating a G9a/GSK-3p-mediated disorder in a subject in need thereof, particularly by inhibiting GSK-3P and G9a; with the proviso that formula (I), or a salt, solvate or stereoisomer thereof, preferably does not contain any one of the following compounds selected from the group consisting of: Ethyl 4-(4-chlorophenyl)-6-methyl-2-oxo-1 , 2,3,4- tetrahydropyrimidine-5-carboxylate; Ethyl 4-(3-chlorophenyl)-6-methyl-2-oxo-1 ,2,3,4- tetrahydropyrimidine-5-carboxylate; Ethyl 6-methyl-2-oxo-4-p-tolyl-1 ,2,3,4- tetrahydropyrimidine-5-carboxylate;
  • the compound of formula (I) is characterized by:
  • R being selected from the group consisting of: H; fluorine; chlorine; (Ci-Cw)haloalkyl; - O-(Ci-C )alkyl; (Ci-Cw)alkyl and -O-(Ci-Cw)haloalkyl;
  • X represents O or S
  • Z represents O or NH
  • R 1 a (Ci-C4)alkyl; or a 4-halophenyl.
  • the compound of formula (I) is characterized by:
  • R being selected from the group consisting of: H; fluorine; chlorine; (Ci-C4)haloalkyl; - O-(Ci-C4)alkyl; (Ci-C4)alkyl and -O-(Ci-C4)haloalkyl;
  • X represents O or S
  • Z represents O or NH
  • R 1 a (Ci-C4)alkyl; or a 4-halophenyl ring.
  • the compound is of formula (II), or a salt, solvate or stereoisomer thereof: wherein:
  • R is one or more radicals independently selected from the group consisting of: H; OH; CN; fluorine; chlorine; (Ci-Cw)haloalkyl; -0-(Ci-Cio)alkyl; (Ci-Cio)alkyl; -O-(Ci- Cw)haloalkyl; SF5; S(O)2R’I; NR ⁇ R’s; CONR ⁇ R’s; and COOR’e;
  • R'i, R'2, R’3, R’4, R’s and R’e are independently selected from the group consisting of: -H, (Ci-Cw)alkyl; (Ci-Cw)haloalkyl; and (C3-C8)cycloalkyl; and
  • X represents O or S; with the proviso that formula (II), or a salt, solvate or stereoisomer thereof, does preferably not contain any one of the following compounds selected from the group consisting of: Ethyl 4-(4- chlorophenyl)-6-methyl-2-oxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate; Ethyl 4-(3- chlorophenyl)-6-methyl-2-oxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate; Ethyl 6-methyl-2- oxo-4-p-tolyl4,2,3,4-tetrahydropyrimidine-5-carboxylate; Ethyl 6-methyl-2-oxo-4-phenyl-
  • R is one or more radicals independently selected from the group consisting of: H; fluorine; chlorine; (Ci-C4)haloalkyl; -O-(Ci-C4)alkyl; (Ci-C4)alkyl and -O-(Ci- C4)haloalkyl.
  • X represents S
  • R is one or more radicals independently selected from the group consisting of: H; Cl; - OCH 3 ; CH 3 and CF 3 .
  • the compound of formula (II) is selected from the group consisting of: a) Ethyl 6-methyl-4-phenyl-2-thioxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate (T1); b) Ethyl 4-(4-chlorophenyl)-6-methyl-2-thioxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate (T2); c) Ethyl 4-(4-methoxyphenyl)-6-methyl-2-thioxo-1 ,2,3,4-tetrahydropyrimidine-5- carboxylate (T3); d) Ethyl 6-methyl-2-thioxo-4-(p-tolyl)-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate (T4); e) Ethyl 4-(3,4-dichlorophenyl)-6-methyl-2-thioxo-1 ,2,3,4-tetrahydr
  • the compound of formula (II) is characterized by:
  • R is one or more radicals independently selected from the group consisting of: H; Cl; - OCH 3 ; CH 3 and CF 3 .
  • the compound of formula (II) is selected from the group consisting of: a) Ethyl 4-(3,4-dichlorophenyl)-6-methyl-2-oxo-1 ,2,3,4-tetrahydropyrimidine-5- carboxylate (U5); b) Ethyl 6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-1 ,2,3,4-tetrahydropyrimidine-5- carboxylate (U6); c) Ethyl 6-methyl-2-oxo-4-(2,4-dichlorophenyl)-1 ,2,3,4-tetrahydropyrimidine-5- carboxylate (U7); and any salt, solvate or stereoisomer thereof.
  • the compound is of formula (III), or a salt, solvate or stereoisomer thereof: wherein:
  • R is one or more radicals independently selected from the group consisting of: H; fluorine; chlorine; (Ci-C4)haloalkyl; -O-(Ci-C4)alkyl; (Ci-C4)alkyl; and -O-(Ci- C4)haloalkyl.
  • R is one or more radicals independently selected from the group consisting of: H; Cl; - OCH 3 ; CH 3 and -OCF 3 .
  • the compound of formula (III) is selected from the group consisting of: a) /V-(4-Chlorophenyl)-6-methyl-2-oxo-4-phenyl-1 ,2,3,4-tetrahydropyrimidine-5- carboxamide (CU1); b) A/, 4-Bis(4-chlorophenyl)-6-methyl-2-oxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxamide (CU2); c) / ⁇ /-(4-Chlorophenyl)-4-(4-methoxyphenyl)-6-methyl-2-oxo-1, 2,3,4- tetrahydropyrimidine-5-carboxamide (CU3); d) /V-(4-Chlorophenyl)-6-methyl-2-oxo-4-(p-tolyl)-1 ,2,3,4-tetrahydropyrimidine-5- carboxamide (CU4); e) / ⁇ /-(4-Chlor
  • the G9a/GSK-3p-mediated disorder is a protein-aggregation disease and the compound is for use in the treatment and/or prevention of a proteinaggregation disease.
  • the protein-aggregation disease is Alzheimer’s disease, Parkinson’s disease or Huntington’s disease.
  • the G9a/GSK-3p-mediated disorder is a disease selected from the group consisting of Alzheimer’s disease, amyotrophic lateral sclerosis, age-related tau astrogliopathy, aortic amyloidosis, argyrophilic grain disease, British familial dementia, cardiac amyloidosis, cerebral amyloid angiopathy, chronic traumatic encephalopathy, corneal dystrophies, corticobasal degeneration, Creutzfeldt-Jakob disease, Danish familial dementia, Down syndrome, familial amyloidosis, familial corneal amyloidosis, fatal insomnia, frontotemporal dementia, Gerstmann-Straussler-Scheinker disease, globular glial tauopathy, hereditary cerebral hemorrhage with amyloidosis, Huntington’s disease, inflammation- associated amyloidosis, kuru, Lewy bodies dementia, Mediterranean fever, Niemann-Pick disease type C, Parkinson’s disease, Pick’s disease
  • any ranges given include both the lower and the upper end-points of the range.
  • the present invention provides the use of compounds of formula (I) (herein referred to as “compounds of the invention”) in preventing or treating an G9a/GSK-3p- mediated disorder in a subject in need thereof, particularly by inhibiting GSK-3P and G9a. or a salt, solvate or stereoisomer thereof; wherein:
  • R is one or more radicals independently selected from the group consisting of: H; OH;
  • R'i, R'2, R’3, R’4, R’ 5 and R’e are independently selected from the group consisting of: -H, (Ci-Cw)alkyl; (Ci-Cw)haloalkyl; and (Cs-Csjcycloalkyl;
  • X represents O or S
  • Z represents O or NH
  • R 1 is selected from the group consisting of: a (Ci-Cw)alkyl; or a 4-halophenyl ring; with the proviso that formula (I), or a salt, solvate or stereoisomer thereof, does preferably not contain any one of the following compounds selected from the group consisting of: Ethyl 4-(4- chlorophenyl)-6-methyl-2-oxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate; Ethyl 4-(3- chlorophenyl)-6-methyl-2-oxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate; Ethyl 6-methyl-2- oxo-4-p-tolyl-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate; Propyl 4-(3-chlorophenyl)-6-methyl-
  • alkyl refers to a straight or branched hydrocarbon chain radical containing no unsaturation, and which is attached to the rest of the molecule by a single bond.
  • Typical alkyl groups have from 1 to about 10, 1 to about 8, or 1 to about 6 carbon atoms, e. g., methyl, ethyl, n-propyl, /-propyl, n-butyl, f-butyl, n-pentyl, etc. If substituted by cycloalkyl, it corresponds to a "cycloalkylalkyl" radical, such as cyclopropyl methyl.
  • aryl If substituted by aryl, it corresponds to an "arylalkyl” radical, such as benzyl, benzhydryl or phenethyl. If substituted by heterocyclyl, it corresponds to a "heterocyclylalkyl” radical.
  • haloalkyl refers to an alkyl group, as defined above, having carbon atoms substituted with at least one halo or halogen groups.
  • the alkyl group contains at least 1 , and at most 10, carbon atoms substituted with at least one halo or halogen group, halogen being as defined herein.
  • C1-C10 haloalkyl groups useful in the present invention include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl and n-butyl substituted independently with one or more halo groups, e.g., fluoro, chloro, bromo and iodo.
  • a particularly useful example of a (C1-C10)haloalkyl group is -CF3. It is noted that preferably, the invention refers to (C1-C6)haloalkyl, (C1-C4)haloalkyl and (C1-C2)haloalkyl groups.
  • the term “-O-(C1-C10)haloalkyl” refers to the group RaO-, where Ra is a haloalkyl, preferably a C1-C10 haloalkyl, as defined above.
  • An exemplary C1-C10 haloalkoxy group useful in the present invention includes, but is not limited to, trifluoromethoxy.
  • the term “-O-(C1-C10)haloalkyl” can thus also be referred to as a C1 to C10 haloalkoxy group.
  • alkoxy refers to a -O-alkyl such as -0-(Ci-Cio)alkyl, where the alkyl group, such as (Ci-Cio)alkyl, is an alkyl as defined above and the term "C1- C10 alkoxy” or “-0-(Ci-Cio)alkyl” refers to the group -O-alkyl , where the alkyl group is a C1- C10 alkyl as defined above.
  • Examples of "-0-(Ci-Cio)alkyl” groups useful in the present invention include, but are not limited to, methoxy, ethoxy, propyloxy, and isopropyloxy.
  • halogen refers to bromo, chloro, iodo or fluoro.
  • salt must be understood as any form of a compound used in accordance with this invention in which said compound is in ionic form or is charged and coupled to a counter-ion (a cation or anion) or is in solution.
  • This definition also includes quaternary ammonium salts and complexes of the active molecule with other molecules and ions, particularly, complexes formed via ionic interactions.
  • the definition includes in particular physiologically acceptable salts; this term must be understood as equivalent to "pharmacologically acceptable salts” or “pharmaceutically acceptable salts”.
  • the term "pharmaceutically acceptable salts” means any salt that is tolerated physiologically (normally meaning that it is not toxic, particularly, as a result of the counter-ion) when used in an appropriate manner for a treatment, applied or used, particularly, in humans and/or mammals.
  • physiologically acceptable salts may be formed with cations or bases and, in the context of this invention, are understood to be salts formed by at least one compound used in accordance with the invention -normally an acid (deprotonated)- such as an anion and at least one physiologically tolerated cation, preferably inorganic, particularly when used in humans and/or mammals.
  • Salts with alkali and alkali earth metals are preferred particularly, as well as those formed with ammonium cations (NH 4 + ).
  • Preferred salts are those formed with (mono) or (di)sodium, (mono) or (di)potassium, magnesium or calcium.
  • These physiologically acceptable salts may also be formed with anions or acids and, in the context of this invention, are understood as being salts formed by at least one compound used in accordance with the invention - normally protonated, for example in nitrogen - such as a cation and at least one physiologically tolerated anion, particularly when used on humans and/or mammals.
  • This definition specifically includes in the context of this invention a salt formed by a physiologically tolerated acid, i.e., salts of a specific active compound with physiologically tolerated organic or inorganic acids - particularly when used on humans and/or mammals.
  • a physiologically tolerated acid i.e., salts of a specific active compound with physiologically tolerated organic or inorganic acids - particularly when used on humans and/or mammals.
  • this type of salts are those formed with: hydrochloric acid, hydrobromic acid, sulphuric acid, methanesulfonic acid, formic acid, acetic acid, oxalic acid, succinic acid, malic acid, tartaric acid, mandelic acid, fumaric acid, lactic acid or citric acid.
  • solvate should be understood as meaning any form a compound in accordance with the invention in which said compound is bonded by a non-covalent bond to another molecule (normally a polar solvent), including especially hydrates and alcoholates, like for example, methanolate.
  • a polar solvent normally a polar solvent
  • a preferred solvate is the hydrate.
  • prodrug is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compounds of the invention.
  • examples of prodrugs include, but are not limited to, derivatives and metabolites of the compounds of formula (I) that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues.
  • prodrugs of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid.
  • the carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule.
  • Prodrugs can typically be prepared using well-known methods.
  • any compound of formula (I) referred to herein is intended to represent such specific compound as well as certain variations or forms.
  • compounds referred to herein may have asymmetric centres and therefore exist in different enantiomeric or diastereomeric forms.
  • any given compound of formula (I) referred to herein is intended to represent any one of a racemate, one or more enantiomeric forms, one or more diastereomeric forms, and mixtures thereof.
  • stereoisomerism or geometric isomerism about the double bond is also possible, therefore in some cases the molecule could exist as (E)-isomer or (Z)-isomer (trans and cis isomers).
  • each double bond will have its own stereoisomerism, that could be the same as, or different to, the stereoisomerism of the other double bonds of the molecule.
  • compounds referred to herein may exist as atropisomers. All the stereoisomers including enantiomers, diastereoisomers, geometric isomers and atropisomers of the compounds referred to herein, and mixtures thereof, are considered within the scope of the present invention.
  • any compound of formula (I) referred to herein may exist as tautomer.
  • tautomer refers to one of two or more structural isomers of a compound that exist in equilibrium and are readily converted from one isomeric form to another.
  • the compound of formula (I) is characterized by R being selected from the group consisting of: H; fluorine; chlorine; (Ci-Cw)haloalkyl; - O-(Ci-C )alkyl; -(Ci-Cw)alkyl and -O-(Ci-Cw)haloalkyl;
  • X represents O or S
  • Z represents O or NH
  • R 1 a (Ci-C4)alkyl; or a 4-halophenyl ring.
  • the compound of formula (I) is characterized by
  • R being selected from the group consisting of: H; fluorine; chlorine; (Ci-C4)haloalkyl; - O-(Ci-C4)alkyl; (Ci-C4)alkyl and -O-(Ci-C4)haloalkyl;
  • X represents O or S
  • Z represents O or NH
  • R 1 a (Ci-C4)alkyl; or a 4-halophenyl ring.
  • the compound of formula (I) is characterized by
  • R being selected from the group consisting of: H; fluorine; chlorine; (Ci-C2)haloalkyl such as CF3 or CHF2; -O-(Ci-C2)alkyl such as a methoxy group; (Ci-C2)alkyl such as a methyl group and -O-(Ci-C2)haloalkyl such as a OCF3;
  • X represents O or S
  • Z represents O or NH
  • R 1 a (Ci-C2)alkyl or a 4-halophenyl ring.
  • the compound is of formula (II), or a salt, solvate or stereoisomer thereof; wherein: R is one or more radicals independently selected from the group consisting of: H; OH; CN; fluorine; chlorine; -(Ci-Cw)haloalkyl; -0-(Ci-Cio)alkyl; -(Ci-Cw)alkyl; -O-(Ci- Cw)haloalkyl; SFs; S(O)2R’I; NR ⁇ R’s; CONR ⁇ R’s; and COOR’e;
  • R'i, R'2, R’3, R’4, R’ 5 and R’e are independently selected from the group consisting of: -H, (Ci-Cw)alkyl; (Ci-Cw)haloalkyl; and (C3-C8)cycloalkyl; and
  • X represents O or S; with the proviso that formula (II), or a salt, solvate or stereoisomer thereof, does preferably not contain any one of the following compounds selected from the group consisting of: Ethyl 4-(4- chlorophenyl)-6-methyl-2-oxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate; Ethyl 4-(3- chlorophenyl)-6-methyl-2-oxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate; Ethyl 6-methyl-2- oxo-4-p-tolyl-l,2,3,4-tetrahydropyrimidine-5-carboxylate; Ethyl 6-methyl-2-oxo-4-phenyl- l,2,3,4-tetrahydropyrimidine-5-carboxylate; Ethyl 6-methyl-2-oxo-4-m-tolyl-1 ,2,3,4 tetrahydropyrimidine-5
  • R is one or more radicals independently selected from the group consisting of: H; fluorine; chlorine; (Ci-C4)haloalkyl; -O-(Ci-C4)alkyl; (Ci-C4)alkyl and -O-(Ci- C4)haloalkyl.
  • R is one or more radicals independently selected from the group consisting of: H; fluorine; chlorine; (Ci-C2)haloalkyl; -O-(Ci-C2)alkyl; (Ci-C2)alkyl and -O-(Ci- C2)haloalkyl.
  • X represents S
  • R is one or more radicals independently selected from the group consisting of: H; Cl; - OCH 3 ; CH 3 and CF 3 . More preferably, wherein the compound of formula (II) is selected from the group consisting of: a) Ethyl 6-methyl-4-phenyl-2-thioxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate (T1); b) Ethyl 4-(4-chlorophenyl)-6-methyl-2-thioxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate (T2); c) Ethyl 4-(4-methoxyphenyl)-6-methyl-2-thioxo-1 ,2,3,4-tetrahydropyrimidine-5- carboxylate (T3); d) Ethyl 6-methyl-2-thioxo-4-(p-tolyl)-1 ,2,3,4-tetrahydropyrimidine-5-carboxylate (T4)
  • the compound is of formula (II) is characterized by:
  • R being one or more radicals independently selected from the group consisting of: H; Cl; -OCH 3 ; CH 3 and CF 3 .
  • the compound of formula (II) is selected from the group consisting of: a) Ethyl 4-(3,4-dichlorophenyl)-6-methyl-2-oxo-1 ,2,3,4-tetrahydropyrimidine-5- carboxylate (U5); b) Ethyl 6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-1 ,2,3,4-tetrahydropyrimidine-5- carboxylate (U6); c) Ethyl 6-methyl-2-oxo-4-(2,4-dichlorophenyl)-1 ,2,3,4-tetrahydropyrimidine-5- carboxylate (U7); and any salt, solvate or stereoisomer thereof.
  • the compound is of formula (III), or a salt, solvate or stereoisomer thereof; wherein:
  • R is one or more radicals independently selected from the group consisting of: H; fluorine; chlorine; -(Ci-C4)haloalkyl; -O-(Ci-C4)alkyl; -(Ci-C4)alkyl; and -O-(Ci- C4)haloalkyl.
  • R is one or more radicals independently selected from the group consisting of: H; Cl; - OCH 3 ; CH 3 and -OCF 3 .
  • the compound of formula (III) is selected from the group consisting of: a) /V-(4-Chlorophenyl)-6-methyl-2-oxo-4-phenyl-1 ,2,3,4-tetrahydropyrimidine-5- carboxamide (CU1); b) A/, 4-Bis(4-chlorophenyl)-6-methyl-2-oxo-1 ,2,3,4-tetrahydropyrimidine-5-carboxamide (CU2); c) / ⁇ /-(4-Chlorophenyl)-4-(4-methoxyphenyl)-6-methyl-2-oxo-1, 2,3,4- tetrahydropyrimidine-5-carboxamide (CU3); d) /V-(4-Chlorophenyl)-6-methyl-2-oxo-4-(p-tolyl)-1 ,2,3,4-tetrahydropyrimidine-5- carboxamide (CU4); e) / ⁇ /-
  • G9a and GLP are major enzymes that catalyze the mono- and dimethylation of a lysine residue at position 9 of histone H3 (H3K9me1 and H3K9me2). These enzymes are also known as EHMT2 and EHMT1 (euchromatin histone-lysine N- methyltransferases 2 and 1).
  • H3K9me2 is an epigenetic mark related to transcriptional repression. G9a and GLP are involved in epigenetic transcriptional repression through H3K9me2. The inhibition of G9a has been widely reported as useful for the control of biological processes, such as cell proliferation and cell differentiation, mediated by transcriptional repression by H3K9me2.
  • Glycogen synthase kinase- 3 (GSK-3) is a serine/threonine kinase widely expressed and active in unstimulated tissues.
  • GSK-3 exists in two isoforms, GSK-3a and GSK- 3p, having 98% homology in their catalytic domain and difference in their C-terminal and N- terminal sequences.
  • the GSK-3P isoform is predominantly expressed in the brain. It is one of the predominant kinases which cause tau hyperphosphorylation, destabilizes the microtubules, and leads to the formation of NFTs.
  • APP and presenilin 1 (PS1) are also GSK- 3p substrates. GSK-3P is therefore implicated in controlling the synthesis of Ap as well.
  • GSK-3P inhibition is said to play a role in reducing the formation of Ap plaques. Inhibition of GSK-3P in cultured neurons also tends to reduce Ap-induced neurotoxicity.
  • the role of Ap has also been hypothesized in developing filaments similar to paired helical filaments (PHF), an important part of NFTs, one of AD's pathogenic characteristics.
  • PHF paired helical filaments
  • the present invention provides compounds which are potent G9a and GSK3P inhibitors.
  • the term “inhibit” refers to a reduction or decrease in a quality or quantity, compared to a baseline.
  • the G9a and/or GSK3p-mediated disorder is associated with overexpression of G9a.
  • G9a overexpression has already been reported in numerous cancers such as hematologic, breast, gastric, ovarian, prostate, lung, colorectal, liver, urinary bladder, and brain cancers has sparked interest in it for therapeutic development (S. H. Barghout, et al., “Chemical biology and pharmacology of histone lysine methylation inhibitors”, Biochim. Biophys. Acta Gene Regul. Meeh. 2022, 1865:194840); as well as involved in immune system activation (S.
  • the compound of formula (I) or any of the compounds described in the present invention can be administered in combination with any other therapeutic agent for preventing or treating the disorder, including G9a and/or GSK3P inhibitors, where the combination of the drugs together is safer or more effective than either drug alone.
  • Such other drug(s) may be administered, by a route and in an amount commonly used therefore, contemporaneously or sequentially with the composition.
  • a pharmaceutical composition in unit dosage form containing such other drugs and the compound of formula I is preferred.
  • the combination therapy may also include therapies in which the compound of Formula I and one or more other drugs are administered on different overlapping schedules.
  • the compounds of the present invention and the other active ingredients may be used in lower doses than when each is used singly.
  • the pharmaceutical compositions of the present invention include those that contain one or more other active ingredients, in addition to a compound of Formula I.
  • the above combinations include combinations of a compound of the present invention not only with one other active compound, but also with two or more other active compounds.
  • the compound of Formula I can be combined with a variety of different anti-neurodegenerative drugs, including those targeting Alzheimer’s disease, Parkinson’s disease and other related conditions.
  • the compound of Formula I can be combined with the following, but not limited to, anticholinergic antiparkinson agents such as diphenhydramine benztropine, trihexyphenidyl, procyclidine or biperiden, dopaminergic antiparkinsonism agents such as ramipexole, rotigotine, selegiline, carbidopa I levodopa, bromocriptine, rasagiline, entacapone, ropinirole, pramipexole or safinamide, or miscellaneous antiparkinson agents such as istradefylline.
  • anticholinergic antiparkinson agents such as diphenhydramine benztropine, trihexyphenidyl, procyclidine or biperiden
  • dopaminergic antiparkinsonism agents such as ramipexole, rotigotine, selegiline, carbidopa I levodopa, bromocriptine, rasagiline, entacap
  • the compound of Formula I can also be combined with the following, but not limited to, atypical antipsychotic agents such as rexpiprazole; cholinesterase inhibitors such as donepezil, galantamine, or rivastigmine; NMDA antagonists such as memantine; or diseasemodifying immunotherapy such as lecanemab.
  • atypical antipsychotic agents such as rexpiprazole
  • cholinesterase inhibitors such as donepezil, galantamine, or rivastigmine
  • NMDA antagonists such as memantine
  • diseasemodifying immunotherapy such as lecanemab.
  • compounds of the present invention may be used in combination with other drugs that are used in the prevention, treatment, control, amelioration, or reduction of risk of the diseases or conditions for which compounds of the present invention are useful as described above.
  • Such other drugs may be administered, by a route and in an amount commonly used therefore, contemporaneously or sequentially with a compound of the present invention.
  • a pharmaceutical composition containing such other drugs in addition to the compound of the present invention is preferred.
  • the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients, in addition to a compound of the present invention.
  • the weight ratio of the compound of the compound of the present invention to the second active ingredient may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, when a compound of the present invention is combined with another agent, the weight ratio of the compound of the present invention to the other agent will generally range from about 1000:1 to about 1 :1000, preferably about 200:1 to about 1 :200. Combinations of a compound of the present invention and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used. In such combinations the compound of the present invention and other active agents may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent to, or subsequent to the administration of other agent(s).
  • the G9a and/or GSK3p-mediated disorder is a protein-aggregation disease and the compound of formula (I) or any of the compounds described in the present invention, are for use in the treatment and/or prevention of a protein-aggregation disease.
  • the protein-aggregation disease is preferably Alzheimer’s disease, Parkinson’s disease or Huntington’s disease, more preferably Alzheimer’s disease.
  • the disease is selected from the group consisting of Alzheimer’s disease, amyotrophic lateral sclerosis, age-related tau astrogliopathy, aortic amyloidosis, argyrophilic grain disease, British familial dementia, cardiac amyloidosis, cerebral amyloid angiopathy, chronic traumatic encephalopathy, corneal dystrophies, corticobasal degeneration, Creutzfeldt-Jakob disease, Danish familial dementia, Down syndrome, familial amyloidosis, familial corneal amyloidosis, fatal insomnia, frontotemporal dementia, Gerstmann-Straussler-Scheinker disease, globular glial tauopathy, hereditary cerebral hemorrhage with amyloidosis, Huntington’s disease, inflammation-associated amyloidosis, kuru, Lewy bodies dementia, Mediterranean fever, Niemann-Pick disease type C, Parkinson’s disease, Pick’s disease, primary age-related tauopathy, progressive subcortical gli
  • the disorder is a cancer, such as lymphoma, leukemia, melanoma, breast cancer, ovarian cancer, hepatocellular carcinoma, prostate carcinoma, lung cancer, brain cancer, or hematological cancer.
  • a cancer such as lymphoma, leukemia, melanoma, breast cancer, ovarian cancer, hepatocellular carcinoma, prostate carcinoma, lung cancer, brain cancer, or hematological cancer.
  • the disorder is an immune-mediated disease.
  • the compounds are used for reducing substance (alcohol) consumption by a subject.
  • the disorder is an imprinting disorder, such as Prader-Willi syndrome, transient neonatal diabetes mellitus, Silver-Russell syndrome, Albright hereditary osteodystrophy, pseudohypoparathyroidism, Birk-Barel mental retardation, Beckwith- Wiedemann syndrome, Temple syndrome, Kagami-Ogata syndrome, Angelman syndrome, precocious puberty, Schaaf-Yang syndrome, sporadic pseudohypoparathyroidism lb, or maternal uniparental disomy of chromosome 20 syndrome.
  • imprinting disorder such as Prader-Willi syndrome, transient neonatal diabetes mellitus, Silver-Russell syndrome, Albright hereditary osteodystrophy, pseudohypoparathyroidism, Birk-Barel mental retardation, Beckwith- Wiedemann syndrome, Temple syndrome, Kagami-Ogata syndrome, Angelman syndrome, precocious puberty, Schaaf-Yang syndrome, sporadic pseudohypopara
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the compounds of the invention (I) as well as solvates, salts or stereoisomers thereof, as defined above. All the embodiments provided above, under the first aspect of the invention, concerning the compounds of formula (I), are also embodiments of the pharmaceutical composition of the invention.
  • terapéuticaally effective amount it is understood the amount of the compound(s) that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed.
  • the precise therapeutic dose of the component(s), as well as the amount of the compound(s) of the invention, may depend on several variables. Some of these would be: route of administration, time of drug release (e.g., instant or extended), administration schedule, pain severity, condition of the patient, and the like.
  • compositions can be prepared as a liquid, semi-solid or solid dosage form, for example in the form of solutions for injection, drops, juices, syrups, sprays, suspensions, tablets, patches, capsules, dressings, suppositories, ointments, creams, lotions, gels, emulsions, aerosols or in multiparticulate form, for example in the form of pills or granules, if appropriate compressed into tablets, decanted into capsules or suspended in a liquid, or administered as such.
  • compositions can be prepared with the aid of conventional means, devices, methods or processes known in the art.
  • Pharmaceutically acceptable adjuvants, vehicles or excipients which may be used in such compositions are adjuvants, vehicles or excipients known to those skilled in the art or commonly used in the preparation of therapeutic compositions, which may be selected, for example, from the group consisting of excipients, fillers, solvents, diluents, surfactants, colorants, preservatives, disintegrants, sliding agents, lubricants, flavoring agents or binders.
  • pharmaceutically acceptable refers to pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio in animals and, particularly, in humans.
  • the term “animal” or “subject” or “patient” shall refer to a vertebrate animal. Such animals include both domestic animals; for example, livestock, laboratory animals and household pets, and non-domestic animals such as wildlife.
  • the animal is a vertebrate.
  • the animal is a domestic mammal or a human.
  • physiologically compatible adjuvants or the number of adjuvants to be used depends on the form of administration of the pharmaceutical composition, i.e., oral, subcutaneous, parenteral, intravenous, intraperitoneal, intradermal, intramuscular, intranasal, buccal, rectal, otic or intratympanic.
  • Preparations in the form of tablets, dragees, capsules, granules, pills, drops, in particular otic drops, juices or syrups are preferably suitable for oral administration; solutions, suspensions, easily reconstitutable dry preparations or also sprays are preferably suitable for parenteral, topical or inhalation administration.
  • the compounds in accordance with the invention used in the pharmaceutical composition in accordance with the invention in a depot, in a dissolved form or in a dressing, or if appropriate having added other agents favoring penetration into the skin, are preparations suitable for percutaneous administration.
  • the preparation forms administrable orally or percutaneously can also release the respective compound according to the invention in a delayed form.
  • the active drug components can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulphate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • suitable binders, lubricants, disintegrating agents, and colouring agents can also be incorporated into the mixture.
  • Suitable binders include starch, gelatine, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
  • Gelatine capsules contain the active ingredient and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • Liquid dosage forms for oral administration can contain colouring and flavouring to increase patient acceptance.
  • the dosage administered of the pharmaceutical composition will, of course, vary depending on the use and known factors such as the age, health, and weight of the recipient; nature and extent of symptoms, concurrent treatments, if any, frequency of treatment, and the effect desired.
  • the recipient may be any type of mammal, but is preferably a human.
  • Thin-layer chromatography was performed with aluminum-backed sheets with silica gel 60 F254 (Merck, ref 1 .05554 or Sigma- Aldrich, ref 60805), and spots were visualized with UV light, 1 % aqueous solution of KMnO4 and/or ninhydrin.
  • HRMS high-resolution mass spectrometry
  • analyses were performed with an LC/MSD TOF Agilent Technologies spectrometer. Analytical grade solvents were used for crystallization, while pure for synthesis solvents were used in the reactions, extractions, and column chromatography.
  • T1, T2, T3, T4, T5, T6, T7, T8, U1, U2, U3, U4, U5, U6, U7, CU1 and CU4 were either commercially available or were synthetized following procedures known in the art (CN101463011A, CN1803777A, W02007105233A2).
  • Ethyl 6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (T1).
  • Ethyl 4-(4-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (U3).
  • Ethyl 6-methyl-2-oxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (U4). IR
  • A/-(4-Chlorophenyl)-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5- carboxamide (CU1).
  • G9a/GSK-3p inhibitors were serially diluted between 5.0 nM and 0.001 nM in 100 % DMSO (Sigma, St. Louis, USA). Then, respective concentrations were subsequently diluted in MilliQ purified water to reach a final concentration ranging between 50 and 0.001 pM in 1 % DMSO in well.
  • the following fluorescent assay was used for determination of the G9a (also called EHMT2, the Vietnamese histone methyltransferase 2) inhibition activity (IC50).
  • G9a activity was measured using the G9a Chemiluminescent Assay Kit (Catalog # 52001 L, BPS Bioscience, San Diego, CA, USA), following manufacturer’s instructions.
  • a comparative control compound UNC0638 which corresponds to 2-cyclohexyl-/V-(1-isopropylpiperidin-4-yl)- 6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-amine which is a well-established G9a inhibitor:
  • G9a and G9a-like are members of the Suv39h subgroup of SET domain-containing molecules, and together they are the key HKMTs for H3K9me1 and H3K9me2. Then, to confirm the selectivity against G9a, we performed the following fluorescent assay for the determination of the GLP inhibition activity (IC50), with substrate and comparative control compound UNC0638, which is a well-established inhibitor with a high potency of G9a and GLP. Unfortunately, the binding to GLP is undesirable in the context of the invention because it gives rise to cytotoxicity.
  • GLP activity was measured using the GLP Chemiluminescent Assay Kit (Catalog #53007 BPS Bioscience, San Diego, CA, USA). The experiment was run per triplicate and are provided in Table 2 below.
  • Table 2 shows the percentage of inhibition of GLP from different assayed compounds (three different experiments in triplicate). In general, the percentages of inhibition at 10 pM are less than 50% and were lower than compared to the reference compound UNC0638. It is noteworthy that T2, T6, and U4 were the compounds that showed the lowest percentage of inhibition for GLP, suggesting that they were the most selective for G9a.
  • GSK-3P inhibition activity was measured using the GSK-3P Chemiluminescent Assay Kit (Catalog #, 79700 BPS Bioscience, San Diego, CA, USA), following the manufacturer’s instructions.
  • the WT C. elegans strain (N2), the transgenics CL2006 (dvls2 [pCL12(unc-54/human Ap peptide 1-42 minigene)+rol-6(su1006)]) and BR5706 (byls193 [rab-3p::F3(delta)K280 + myo- 2p::mCherry]; bklslO [aex-3p::hTau V337M + myo-2p::GFP]) provided by the C. elegans Genetic Center were used. Standard methods were used for culturing and observing C. elegans.
  • N2 were propagated at 20°C, while CL2006 and BR5706 worms were maintained at 16°C in a temperature-controlled incubator on a solid nematode growth medium (NGM) seeded with Escherichia coli (E. coli) OP50 (Carolina Biological) strain as a food source.
  • NMM solid nematode growth medium
  • Locomotion assay of G9a/GSK-3p inhibitors was assessed to obtain dose-response profile, evaluating the impact of the pharmacology treatment in motor dysfunction presented by the transgenic strains CL2006.
  • Worms were grown with continuous shaking at 180 rpm at 20 °C for 4 days.
  • Each well contained a final volume of 60 pL, comprising 25- 30 animals in the larva 1 (L1) stage diluted in S-medium solution, G9a inhibitor at the appropriate dose (means that the working solution is 2.4-fold more concentrated than the final concentration in the well), and OP50 (Carolina Biological) inactivated by freeze-thaw cycles suspended in S-medium complete solution to a final OD595 of 0.9 measured in the microplate reader.
  • worms were transferred from the 96-well plates onto an unseeded NGM plate for 45 minutes before starting the trial, allowing the plates to dry.
  • Statistical analysis ordinary One-way ANOVA, post- hoc test Dunnett, *p ⁇ 0.05, **p ⁇ 0.01 , ***p ⁇ 0.001 , ****p ⁇ 0.0001.
  • Table 6 shows that the T2 reduced AB aggregation by approximately 56% compared to the vehicle group, being more effective in reducing AB aggregation than well-established UNC0638 and even UNC0638 in combination with Tideglusib, both of which only reduced AB deposition by approximately 37%.
  • EXAMPLE 3 Activity on mouse model of Alzheimer disease
  • Senescence-accelerated mice prone 8 is a model of age-related neurodegeneration underlying LOAD.
  • This model established through phenotypic selection from AKR/J mice, presents AD-like cognitive symptoms and behavioural abnormalities, including anxiety-like behaviour and depression.
  • SAMP8 brains are characterized by pathological signatures of AD, including neuroinflammation, synaptic deficits, oxidative stress and aberrant epigenetic dysregulation (C. Grinan-Ferre, et. al., “Understanding Epigenetics in the Neurodegeneration of Alzheimer's Disease: SAMP8 Mouse Model”, J Alzheimers Dis. 2018, 62, 943-963).
  • vehicle (20% w/v, (2-hydroxypropyl)-p- cyclodextrin)
  • the test compound was dissolved in 20% w/v (2-hydroxypropyl)-p-cyclodextrin, and the volume of injection was calculated according to the animal weight to reach the precise daily dose.
  • mice were place 90-degree, two-arm, 25-cm-long, 20-cm-high maze of black polyvinyl chloride. Light intensity in the middle of the field was 30 lux.
  • mice were individually habituated to the apparatus for 10 min per day for 3 days. On day 4, the animals were allowed to freely explore two identical objects (A and A or B and B) placed at the end of each arm for a 10 min acquisition trial (first trial-familiarization). Then, a 10-min retention trial (second trial) was carried out 2 h (short-term memory) or 24 h (long-term memory) later. During the Short-term memory retention test, the times that the animal spent exploring the new object (TN) and the old object (TO) was recorded.
  • TN new object
  • TO old object
  • mice Twenty-four hours after the acquisition trial, the mice were tested again, with a new object and an object identical to the new one in the previous trial (A and C, or B and C).
  • TN and TO were measured from the video recordings from each trial session.
  • a Discrimination index (DI) was defined as (TN-TO)/(TN+TO). The maze, the surface, and the objects were cleaned with 70% ethanol between the animals’ trials to eliminate olfactory cues.
  • the learning and memory paradigm is based on the spontaneous exploratory activity of rodents and does not involve rule learning or reinforcement.
  • the object recognition paradigm has been shown to be sensitive to the effects of aging and cholinergic dysfunction, among others (C. Scali, et al., “Nerve growth factor increases extracellular acetylcholine levels in the parietal cortex and hippocampus of aged rats and restores object recognition”, Neurosci Lett. 1994, 170, 117-20; L. Bartolini, et al., “Aniracetam restores object recognition impaired by age, scopolamine, and nucleus basalis lesions”, Pharmacol. Biochem. Behav. 1996, 53, 277-83).
  • Table 7 above shows working memory results by using Novel object recognition test after G9a inhibition treatment with our candidate T2. Interestingly, we showed a significant improvement in SAMP8 treated with T2 in comparison with SAMP8 Control in short- and long-term memories, suggesting the G9a participation of cognitive impairment in Alzheimer’s Disease.
  • mice were euthanized by cervical dislocation. Brains were immediately removed from the skull. Cortex and hippocampus were then isolated and frozen on powdered dry ice. They were maintained at -80 °C for biochemical experiments. For the Golgi staining protocol, see the procedure in the section “Spine density and Golgi staining protocol”.
  • Histone extraction was performed following the manufacturer’s instructions using hippocampal tissue from each experimental group (EpiQuik Total Histone Extraction HT Kit, EpiGentek, #GP-0007-192). The samples were resolved in a 14% SDS-gel, as previously described. [12] To capture chemiluminescence signals were used Amersham Imager 680 and Western blot quantifications were performed using ImageLab software (Bio-Rad). Immunoblots were probed with anti-H3 K9me2 (1 :1000) (Epigentek, #A-4035), and anti-H3 total signaling, #9715).
  • Table 9 shows that the pharmacological treatment with T2 significantly decreases Tau phosphorylation at both positions (Ser 396, and Thr 217) in SAMP8 compared to the control group.
  • Table 10 shows that the pharmacological treatment with T2 significantly increase the dendritic spine density, and the total dendritic length in SAMP8 in comparison with the control group.

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Abstract

La présente invention concerne des inhibiteurs de G9a/GSK-3β puissants et spécifiques, des compositions pharmaceutiques comprenant ces composés, et leurs utilisations dans le traitement ou la prévention de maladies ou d'états liés à la surexpression d'inhibiteurs de G9a/GSK-3β.
PCT/EP2025/059782 2024-04-10 2025-04-09 Inhibiteurs doubles de g9a/gsk-3ss Pending WO2025215108A1 (fr)

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