Heterotricyclic derivatives for the treatment of neurodegenerative diseases

Abstract

The application relates to 8 specific heterotricyclic compounds related to naturally occurring flavonoids such as 7,8-dihydroxyflavone (7,8-DHF). The compounds are useful as pharmaceuticals for the treatment of neurodegenerative diseases.

Description

NOVEL COMPOUNDS AND USES THEREOF

[0001] The present disclosure is related to and claims benefit of GB2418266.9 filed on 12 December 2024, GB2508885.7 filed on 5 June 2025 and GB2516852.7 filed on 9 October 2025 the contents of which are hereby incorporated by reference in their entirety.FIELD OF THE INVENTION

[0002] The present invention relates to novel synthetic compounds related to naturally occurring flavonoids such as 7,8-dihydroxyflavone (7,8-DHF). The invention further relates to the method of synthesis, the use of these compounds as research tools and their use as pharmaceuticals.BACKGROUND TO THE INVENTION

[0003] Flavonoids are a large class of plant secondary metabolites, and they are also common polyphenols in the human diet. Studies have shown that flavonoids have various pharmacological activities, such as anti-tumor, anti-inflammatory, and antioxidant properties. The flavonoid, 7,8-dihydroxyflavone (7,8-DHF), is a naturally occurring flavone found in Godmania aesculifolio, Tridax procumbens and Primula helleri leaves. It is known to act as a potent and selective agonist of tropomyosin receptor kinase B (TrkB), which is the main signaling receptor of neurotrophin brain-derived neurotrophic factor (BDNF).

[0004] 7,8-DHF has been shown to have therapeutic efficacy in several animal models including depression, Alzheimer’s disease, cognitive deficits in schizophrenia, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, traumatic brain injury, cerebral ischemia, fragile X syndrome and Rett syndrome.

[0005] A derivative of 7,8-DHF, 4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8- d]imidazol-8- yl)benzonitrile, also known as CF3CN has been shown to be useful in the treatment of various diseases and conditions including neurodegenerative diseases and movement disorders.

[0006] There is a need for the development of improved flavonoid-like compounds, as well as compositions and therapeutic uses thereof.BRIEF SUMMARY OF THE INVENTION

[0007] At its most general, the present invention relates to novel synthetic flavonoid compounds.

[0008] Compounds were tested in a functional assays and an in vivo animal study to determine the compounds efficacy and potential for use as medicaments.

[0009] In a first aspect of the present invention there is provided a compound as defined by any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof.

[0010] In a second aspect of the present invention there is provided a pharmaceutical composition comprising a compound of any one of the compounds numbered 1 to 8 in Table 1 , or a salt thereof, together with one or more ingredients selected from carriers, diluents, excipients, adjuvants, fillers, buffers, binders, disintegrants, preservatives, antioxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.

[0011] In a third aspect of the present invention there is provided a compound of any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof or a pharmaceutical composition comprising the compound any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof, for use as a medicament.

[0012] In a fourth aspect of the present invention there is provided a method of treatment comprising administering to a subject in need of treatment a therapeutically effective amount of a compound of any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof.

[0013] In a fifth aspect of the present invention there is provided a method of synthesizing a compound of any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof.

[0014] In a sixth aspect of the present invention there is provided an intermediate formed in the method of synthesis of the compound of any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof.

[0015] These and other aspects and embodiments of the invention are described in further detail below.BRIEF SUMMARY OF THE DRAWINGS

[0016] The present invention is described with reference to the figure listed below:

[0017] Figure 1 details the effect of 1 hour pre-treatment with Compound 4 on primary mesencephalic cells injured with aSyn: survival (A), neurite network (B), aSyn aggregation (C) and number of nuclei (D).

[0018] Figure 2 details the pTrkB / TrkB area in protein lysate from primary cortical neurons.

[0019] Figure 3 details a representative image of automated morphological analysis.

[0020] Figure 4 details the neuritogenesis analysis for Plate 1. (A) number of MAP2(+) neurons; (B) neurite network of MAP2(+) neurons; (C) total roots I branching points; and (D) total neurite extremities.

[0021] Figure 5 details the neuritogenesis analysis for Plate 2. (A) number of MAP2(+) neurons; (B) neurite network of MAP2(+) neurons; (C) total roots I branching points; and (D) total neurite extremities.

[0022] Figure 6 details the neuritogenesis analysis for Plate 3. (A) number of MAP2(+) neurons; (B) neurite network of MAP2(+) neurons; (C) total roots I branching points; and (D) total neurite extremities.

[0023] Figure 7 details the neuritogenesis analysis for Plate 4. (A) number of MAP2(+) neurons; (B) neurite network of MAP2(+) neurons; (C) total roots I branching points; and (D) total neurite extremities.

[0024] Figure 8 details the neuritogenesis analysis for Plate 5. (A) number of MAP2(+) neurons; (B) neurite network of MAP2(+) neurons; (C) total roots I branching points; and (D) total neurite extremities.

[0025] Figure 9 details the neuritogenesis analysis for Plate 6. (A) number of MAP2(+) neurons; (B) neurite network of MAP2(+) neurons; (C) total roots I branching points; and (D) total neurite extremities.

[0026] Figure 10 details the neuritogenesis analysis for Plate 7. (A) number of MAP2(+) neurons; (B) neurite network of MAP2(+) neurons; (C) total roots I branching points; and (D) total neurite extremities.

[0027] Figure 11 details the maximal percentage increase in neurite network length for test compounds.

[0028] Figure 12 details the concentration-response curves used to generate ECso values for test compounds.

[0029] Figure 13 details the Social Interaction Test (SIT) - Three Chamber Test, Day 32: Sociability preference index of mice following treatment with test article or positive control.

[0030] Figure 14 details the Social Interaction Test (SIT) - Three Chamber Test, Day 32: Social novelty preference index of mice following treatment with test article or positive control.

[0031] Figure 15 details the Forced Swimming Test (SIT) - Day 33: Immobility of mice following treatment with test article or positive control.DEFINITIONS

[0032] “Alkyl” refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups. An alkyl group may contain from one to twelve carbon atoms (e.g., C1-12 alkyl), such as one to eight carbon atoms (C1-8 alkyl) or one to six carbon atoms (C1-6 alkyl). Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl, and decyl. An alkyl group is attached to the rest of the molecule by a single bond. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more substituents such as those substituents described herein.

[0033] “Haloalkyl” refers to an alkyl group that is substituted by one or more halogens. Exemplary haloalkyl groups include trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1 ,2-difluoroethyl, 3-bromo-2-fluoropropyl, and 1 ,2-dibromoethyl.

[0034] The term “cycloalkyl” employed alone or in combination with other terms, refers to a nonaromatic hydrocarbon ring system (monocyclic, bicyclic, or polycyclic), including cyclized alkyl and alkenyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g. having 2, 3, or 4 fused rings) groups and spirocycles. Cycloalkyl groups can have 3, 4, 5, 6, or 7 ring-forming carbons (C3 - C7). In some embodiments, the cycloalkyl group has 3 to 6 ring members, 3 to 5 ring members, or 3 to 4 ring members. In some embodiments, the cycloalkyl group is monocyclic. In some embodiments, the cycloalkyl group is bicyclic or spirocyclic. Ring-forming carbon atoms of a cycloalkyl group can be optionally oxidized to form an oxo or sulfido group. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e. having a bond in common with) to the cycloalkyl ring, e.g. benzo or thienyl derivatives of cyclopentane, cyclohexane, and the like. A cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclohexenyl, and cyclohexadienyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, nonane andadamantane. Spirocyclic cycloalkyl groups include, but are not limited to spiro-, heptane, octane, nonane, decane and undecane.

[0035] The term " heterocycloalkyl” employed alone or in combination with other terms, refers to both aromatic and non-aromatic rings or ring systems, which may optionally contain one or more carbon-carbon double bonds as part of the ring structure, which has at least one heteroatom ring member independently selected from nitrogen, sulfur, oxygen, and phosphorus, and which has 3 - 12 total ring members. Any heterocycloalkyl ring member may be further mono-, or di-, alkylated (C1-C6) if allowed by rules for carbon valency. Included within the term "heterocycloalkyl” are monocyclic 3-, 4-, 5-, 6-, and 7-, membered heterocycloalkyl groups. Heterocycloalkyl groups can include bicyclic (e.g. having two fused or bridged rings) or spirocyclic (e.g. having two ring systems fused by a single shared carbon atom) ring systems containing 3-12 total ring members. In some embodiments, the heterocycloalkyl group is a monocyclic group having 1 , 2, or 3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heterocycloalkyl group is a poly-, or spirocyclic group having 1 , 2, or 3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. Ringforming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally oxidized to form an oxo or sulfido group or other oxidized linkage (e.g. C (O), S (O), C (S) or S (O)2, N- oxide etc.) or a nitrogen atom can be quaternized. Where a heteroatom is selected to be a nitrogen, this heteroatom may be further functionalized to the corresponding N-alkyl(C1-C6), N- fluoroalkyl (C1-C6) or N-CO-Alkyl(C1-C6). The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e. having a bond in common with) to the heterocycloalkyl ring, e.g. benzo or thienyl derivatives of piperidine, morpholine, azepine, etc. A heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. Examples of monocyclic heterocycloalkyl groups include, but are not limited to oxirane, aziridine, azetidine, pyrrolidine, piperidine, piperizine, pyridine, pyrimidine, pyridazine, pyrazole, pyrrole, imidazole, morpholine, dioxane, pyran, furan, thiophene, thoiazole, and oxazole in any variation of saturation. Examples of polycyclic heterocycloalkyl groups include, but are not limited to, quinoline, isoquinoline, indole, benzofuran, benzimidazole, chromane, chromene, and coumarine. Examples of spirocyclic heterocycloalkyls include, but are not limited to, 1-oxa-8-azaspiro[4.5]decane, 2,2-dimethyl-1-oxa-8-azaspiro[4.5]decane, 1 ,1-dimethyl-2-oxa-8- azaspiro[4.5]decane 3-oxa-9-azaspiro[5.5]undecane, 2,6-diazaspiro[3.4]octan-5-one

[0036] “Alkenyl” refers to substituted or unsubstituted hydrocarbon groups, including straightchain or branched-chain alkenyl groups containing at least one double bond. An alkenyl group may contain from two to twelve carbon atoms (e.g., C2-12 alkenyl). Exemplary alkenyl groups include ethenyl ( / .e., vinyl), prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1 , 4-dienyl, and the like. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted by one or more substituents such as those substituents described herein.

[0037] “Alkynyl” refers to substituted or unsubstituted hydrocarbon groups, including straightchain or branched-chain alkynyl groups containing at least one triple bond. An alkynyl group may contain from two to twelve carbon atoms (e.g., C2-12 alkynyl). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted by one or more substituents such as those substituents described herein.

[0038] “Aryl” refers to an all carbon monocyclic or fused-ring polycyclic (i.e. , rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi electron system. Examples, without limitation, of aryl groups are phenyl, napthalenyl and anthracenyl. The aryl group may be substituted or unsubstituted.

[0039] “Ester” refers to a functional group -COO and may also be referred to as an “ester link”. Esters are formed by the condensation reaction between an alcohol and a carboxylic acid.

[0040] The term “halo” or, alternatively, “halogen” means fluoro or fluorine, chloro or chlorine, bromo or bromine and iodo or iodine.

[0041] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons or heteroatoms of the structure. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogensubstituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocycle, an aralkyl, a carbocycle, a heterocycle, a cycloalkyl, a heterocycloalkyl, an aromatic and heteroaromatic moiety.

[0042] It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted,” references to chemical moieties herein are understood to include substituted variants. For example, reference to a “heteroaryl” group or moiety implicitly includes both substituted and unsubstituted variants.

[0043] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0044] “Optional” or “optionally” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, “optionally substituted aryl” means that the aryl group may or may not be substituted and that the description includes both substituted aryl groups and aryl groups having no substitution.

[0045] Compounds of the present disclosure also include crystalline and amorphous forms of those compounds, pharmaceutically acceptable salts, and active metabolites of these compounds having the same type of activity, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as mixtures thereof.

[0046] The compounds described herein may exhibit their natural isotopic abundance, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure. For example, hydrogen has three naturally occurring isotopes, denoted1H (protium),2H (deuterium), and3H (tritium). Protium is the most abundant isotope of hydrogen in nature.Enriching deuterium may afford certain therapeutic advantages, such as increased in vivo halflife and / or exposure, or may provide a compound useful for investigating in vivo routes of drug elimination and metabolism. Isotopically enriched compounds may be prepared by conventional techniques well known to those skilled in the art.

[0047] “Isomers” are different compounds that have the same molecular formula. “Regioisomers” are isomers that differ only in the attachment point of a single functional group. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1 :1 mixture of a pair of enantiomers is a “racemic” mixture. The term “(±)” is used to designate a racemic mixture where appropriate. “Diastereoisomers” or “diastereomers” are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. The absolute stereochemistry is specified according to the Cahn-lngold-Prelog R-S system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S. Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) in which they rotate plane polarized light at the wavelength of the sodium D line. Certain compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, the asymmetric centers of which can be defined, in terms of absolute stereochemistry, as (R)- or (S)-. The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible stereoisomers, including racemic mixtures, optically pure forms, mixtures of diastereomers and intermediate mixtures. Optically active (R)- and (S)-isomers can be prepared using chiral synthons or chiral reagents or resolved using conventional techniques. The optical activity of a compound can be analyzed via any suitable method, including but not limited to chiral chromatography and polarimetry, and the degree of predominance of one stereoisomer over the other isomer can be determined.

[0048] Chemical entities having carbon-carbon double bonds or carbon-nitrogen double bonds may exist in Z- or E- form (or cis- or trans- form). Furthermore, some chemical entities may exist in various tautomeric forms. Unless otherwise specified, chemical entities described herein are intended to include all Z-, E- and tautomeric forms as well.

[0049] Isolation and purification of the chemical entities and intermediates described herein can be affected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layerchromatography or thick-layer chromatography, or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the examples herein below. However, other equivalent separation or isolation procedures can also be used.

[0050] When stereochemistry is not specified, certain small molecules described herein include, but are not limited to, when possible, their isomers, such as enantiomers and diastereomers, mixtures of enantiomers, including racemates, mixtures of diastereomers, and other mixtures thereof, to the extent they can be made by one of ordinary skill in the art by routine experimentation. In those situations, the single enantiomers or diastereomers, i.e., optically active forms, can be obtained by asymmetric synthesis or by resolution of the racemates or mixtures of diastereomers. Resolution of the racemates or mixtures of diastereomers, if possible, can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral high- pressure liquid chromatography (HPLC) column. Furthermore, a mixture of two enantiomers enriched in one of the two can be purified to provide further optically enriched form of the major enantiomer by recrystallization and / or trituration. In addition, such certain small molecules include Z- and E- forms (or cis- and trans- forms) of certain small molecules with carbon-carbon double bonds or carbon-nitrogen double bonds. Where certain small molecules described herein exist in various tautomeric forms, the term “certain small molecule” is intended to include all tautomeric forms of the certain small molecule.

[0051] The term “salt” or “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic 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. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substitutedamines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.

[0052] The phrase “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0053] As used herein, “treatment” or “treating” refers to an approach for obtaining beneficial or desired results with respect to a disease, disorder, or medical condition including but not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit can include, for example, the eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit can include, for example, the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. In certain embodiments, for prophylactic benefit, the compositions are administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.DETAILED DESCRIPTION OF THE INVENTION

[0054] Throughout this disclosure, various patents, patent applications and publications are referenced. The disclosures of these patents, patent applications and publications in their entirety are incorporated into this disclosure by reference for all purposes in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. This disclosure will govern in the instance that there is any inconsistency between the patents, patent applications and publications cited and this disclosure.

[0055] Modifying tropoflavin compounds can significantly change their chemical and biological properties. Such chemical functional groups can include a polar moiety, a monosaccharide, disaccharide, a carbohydrate, an amino acid, an acyl group, a diacid group, and other chemical moieties. For biological systems, the addition of such modifying functional groups can significantly alter the resulting biological activity or tissue targeting. For the end use of these compounds, modifications have major impacts on downstream formulations, preparations, pharmacokinetics, pharmacodynamics, and ultimate end uses.Compounds

[0056] In embodiments, provided herein is one or more pharmaceutically acceptable salts of a compound selected from Table 1.Table 1. CompoundsCompositions

[0057] The compounds described herein may be formulated as a pharmaceutical composition. A pharmaceutical composition may comprise a compound as detailed in Table 1 , or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

[0058] In embodiments, a pharmaceutically acceptable carrier includes a pharmaceutically acceptable excipient, binder, and / or diluent. In embodiments, suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone. In embodiments, suitable pharmaceutically acceptable carriers include, but are not limited to, inert solid fillers or diluents and sterile aqueous or organic solutions. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers, diluents, or excipients are well- known to those in the art. (See, e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995).) Formulations can further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.

[0059] Pharmaceutical compositions comprising a compound as detailed in Table 1 or a pharmaceutically acceptable salt thereof, may also contain one or more additional ingredients including, but not limited to, a mucoadhesive compound, a buffering agent, a plasticizing agent,a stabilizing agent, a taste-masking agent, a flavoring agent, a coloring agent, an antiseptic, an inert filler agent, a preservative, and combinations thereof.

[0060] In embodiments, the formulations may comprise one or more solubilizing agents that increase the solubility of active compounds in the formulation. Suitable solubilizing agents include, for example, complexing agents, surfactants, and the like. Suitable complexing agents include unsubstituted cyclodextrins (such as alpha-cyclodextrin, beta-cyclodextrin) and substituted cyclodextrins, (such as hydroxypropyl beta-cyclodextrin, sulfobutylether-beta- cyclodextrin). Suitable surfactants include polyoxyethylene sorbitan monolaurate (for example, Tween 20), polyoxyethylene sorbitans monooleate (for example, Tween 80), polyethylene glycol (15)-hydroxystearate (for example, Kolliphor® HS 15), PEG-35 castor oil (for example, Kolliphor® EL) and PEG-60 hydrogenated castor oil (for example, Cremophor® RH 60).

[0061] In embodiments, the formulations comprise one or more buffer agents that maintain the pH of the IV solution within a pharmaceutically acceptable range. In certain embodiments, the buffer maintains the pH of the IV solution between about 5 and 9. In specific embodiments, the buffer maintains the pH of the IV solution at about 7.4. Suitable buffers include, for example, citrates, lactate, acetate, maleate, phosphates, and the like. In embodiments, the formulations comprise one or more density modifiers that are used to control the density of the IV formulation. Suitable density modifiers include, for example, dextrose. In embodiments, the formulations comprise one or more isotonicity modifiers that provide a formulation that is iso- osmotic with tissue to prevent pain and irritation when the formulation is administered. Suitable isotonicity modifiers include, for example, electrolytes, monosaccharides, and disaccharides. Examples of isotonicity modifiers include glycerin, dextrose, potassium chloride, and sodium chloride.

[0062] In embodiments, the formulations comprise one or more viscosity enhancers. Suitable viscosity enhancers include, for example, povidone, hydroxyethylcellulose, polyvinyl alcohol, and carbomer (such as, acrylic acid homopolymers and acrylic acid copolymers).

[0063] In embodiments, the formulations comprise one or more preservatives that increase the stability of active compounds in the formulation and / or provide antimicrobial activity. Suitable preservatives include, for example, antimicrobial agents and antioxidants. Examples of antimicrobial agents include benzyl alcohol, methyl paraben, propyl paraben, phenol, cresol, methyl paraben, chlorbutanol, sodium metabisulphite, sodium bisulphite, benzethonium chloride, and benzalkonium chloride. Examples of antioxidants include sodium bisulphite andother sulfurous acid salts, ascorbic acid, salts of ethylenediaminetetraacetic acid (including sodium), alpha tocopherol, butylated hydroxyl hydroxytoluene, and butylated hydroxyanisole.

[0064] A pharmaceutical composition comprising a compound as detailed in Table 1 , or a pharmaceutically acceptable salt thereof; may be formulated in a dosage form selected from the group consisting of: an oral unit dosage form, an intravenous unit dosage form, an intranasal unit dosage form, a suppository unit dosage form, an intradermal unit dosage form, an intramuscular unit dosage form, an intraperitoneal unit dosage form, a subcutaneous unit dosage form, an epidural unit dosage form, a sublingual unit dosage form, a liquid, a lozenge, a fast disintegrating tablet, a lyophilized preparation, a film, a spray (including a nasal spray, an oral spray, or a topical spray), or a mucoadhesive. The oral unit dosage form may be selected from the group consisting of: tablets, pills, pellets, capsules, powders, lozenges, granules, solutions, suspensions, emulsions, syrups, elixirs, sustained-release formulations, aerosols, and sprays. In embodiments, the compounds are formulated as a liquid, a lozenge, a fastdisintegrating tablet, a lyophilized preparation, a film, a spray, or a mucoadhesive.

[0065] The compounds as detailed in Table 1 , or a pharmaceutically acceptable salt thereof can be administered to subjects by a variety of administration modes, including, for example, by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, and oral routes of administration. For prevention and treatment purposes, a compound as detailed in Table 1 , or a pharmaceutically acceptable salt thereof can be administered to a subject in a single bolus delivery, via continuous delivery (e.g., continuous transdermal delivery) over an extended time period, or in a repeated administration protocol (e.g., on an hourly, daily, weekly, or monthly basis).

[0066] Pharmaceutical compositions comprising a compound as detailed in Table 1 , or a pharmaceutically acceptable salt thereof can be supplied as a kit comprising a container that comprises the pharmaceutical composition as described herein. A pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a pharmaceutical composition. Such a kit can further comprise written information on indications and usage of the pharmaceutical composition.Therapeutic Use

[0067] The compounds of the invention, as detailed in Table 1 , have demonstrated efficacy in several in vitro assays. Therefore, the novel synthetic flavonoid compounds of the invention may be useful in the treatment or prevention of medical conditions. More specifically the activity of these compounds is likely to enable their use in the treatment or prevention of medical conditions associated with neurodegeneration and I or neuropsychiatric disorders.

[0068] Further these compounds have been shown to have efficacy in an in vivo mouse model further demonstrating their usefulness as medicaments.WORKED EXAMPLES

[0069] The compounds of the present invention were synthesized using the experimental details described in Example 1.

[0070] Reaction products can be purified by known methods including silica gel chromatography using various organic solvents such as hexane, dichloromethane, ethyl acetate, MeOH and the like, preparative reverse phase high pressure liquid chromatography, or preparative supercritical fluid chromatography.

[0071] Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene and Wuts, Protective Groups in Organic Synthesis, 44th. Ed., Wiley & Sons, 2006, as well as Jerry March, Advanced Organic Chemistry, 4th edition, John Wiley & Sons, publisher, New York, 1992 which are incorporated herein by reference in their entirety.

[0072] Examples 2 to 8 describe the efficacy of these compounds in in vitro assays and an in vivo mouse model.EXAMPLE 1 : REACTION METHODOLOGIES

[0073] In order to prepare the compounds of Formula I and Formula II as exemplified by the compounds 1 to 8 in Table 1 , the following reaction methodologies were employed.AbbreviationsGeneral InformationSolvents and Reagents

[0074] Organic Solvents and chemical reagents were purchased from commercial sources and used as received unless otherwise stated.Preparation of Intermediates

[0075] Known synthetic intermediates were procured from commercial vendors or were obtained using published literature procedures with reference to original published literature as annotated. Additional intermediates were prepared by the representative synthetic processes described herein.Analytical MethodsNuclear Magnetic Resonance

[0076] NMR spectra were recorded using a Bruker 400MHz Avance Neo spectrometer fitted with a Bruker 5mm iProbe, or a Bruker 500MHz Avance III HD spectrometer equipped with a Bruker 5mm SmartProbe™. Spectra were measured at 298 K, unless indicated otherwise, and were referenced relative to the solvent resonance. The chemical shifts are reported in parts per million. Data were acquired using Bruker TopSpin software and processed using MestreNova software.Mass Spectrometry (MS)

[0077] Compounds were analysed by Ultra High Performance Liquid Chromatography Mass Spectroscopy (UPLC) or Liquid Chromatography Mass Spectrometry (LCMS) as indicated. All UPLC / LCMS methods were 3 minute runs.Method 1 - UPLC Acidic MethodApparatus: Waters HCIass; Binary Solvent Pump, SM-FTN, CMA, PDA, QDaColumn: ACQUITY UPLCO CSH C18, 130A, 1.7 pm, 2.1 x 30 mm at 40 °CDetection: UV at 210-400 nm unless otherwise indicated, MS by electrospray ionisationSolvents: A: 0.1 % Formic in water, B: MeCNGradient:Method 2 - LCMS Acidic MethodApparatus: Agilent 1260; Binary Pump, HiP Sampler, Column Compartment, DAD:, G6150 MSColumn: Waters Cortecs C18, 30 x 2.1 mm, 2.7pm, at 40 °CDetection: UV at 260nm + / - 90nm unless otherwise indicated, MS by electrospray ionisationSolvents: A: 0.1 % formic acid in water, B: MeCNGradient:Method 3 - LCMS Method 3Apparatus: Agilent 1260; Binary Pump, HiP Sampler, Column Compartment, DAD:, G6150 MSColumn: Phenomenex Evo C18, 30 x 2.1 mm, 2.6pm, at 40 °CDetection: UV at 260nm + / - 90nm unless otherwise indicated, MS by electrospray ionisationSolvents: A: 0.1 % Ammonia in water, B: MeCNGradient:Compounds 1 & 23,4-difluorophenyl acetate

[0078] Acetyl chloride (34.5 mL, 99% Wt, 1.25 Eq, 480 mmol) was added dropwise to a solution of 3,4-difluorophenol (50.0 g, 1 Eq, 384 mmol) in DCM (1.0 L) and triethylamine (67.0 mL, 1.25 Eq, 480 mmol) at 0 °C. The reaction was allowed to warm to rt and stirred overnight. The reaction was filtered and the precipitate washed with DCM (100 mL). The filtrate was reduced toapproximately half volume, the resulting precipitate was filtered again and the filtrate was dried directly onto Celite and purified by chromatography on silica gel using a gradient of 0-20% MTBE / isohexane to afford 3,4-difluorophenyl acetate (64.85 g, 0.37 mol, 97 %) as a clear colourless oil.

[0079] 1H NMR (500 MHz, DMSO) 5 7.49 (dt, J = 10.8, 9.2 Hz, 1 H), 7.38 (ddd, J = 11.6, 6.9, 2.8 Hz, 1 H), 7.07 - 6.99 (m, 1 H), 2.26 (s, 3H).19F NMR (471 MHz, DMSO) 5 -136.15 (d, J = 22.4 Hz), -142.04 (d, J = 22.3 Hz). MS: The product was analysed by LCMS (Method 2): m / z does not ionise [M+H]+(ES+); does not ionise [M-H]' (ES-), at 1.24 min, 99% purity at 210-400nm.1-(4,5-difluoro-2-hydroxyphenyl)ethan-1-one

[0080] 3,4-difluorophenyl acetate (10.0 g, 99% Wt, 1 Eq, 57.5 mmol) was dissolved in DCM (120 mL) before addition of aluminium chloride (23.0 g, 3 Eq, 173 mmol). The resulting suspension was stirred at rt for 1 h, the solvent was removed in vacuo and the residue was heated to 140 °C for 90 min. The reaction was allowed to cool and aq. HCI (6M) (180 mL) was added cautiously to the residue. The resulting suspension was stirred for 10 min, the solid was filtered off and dried in vacuo to afford 1-(4,5-difluoro-2-hydroxyphenyl)ethan-1-one as a light brown solid. The procedure was repeated with 3,4-difluorophenyl acetate (10.0 g, 57.5 mmol) and the products were combined to afford 1-(4,5-difluoro-2-hydroxyphenyl)ethan-1-one (18.23 g, 0.10 mol, 91 %) as a light brown solid.

[0081] 1H NMR (500 MHz, DMSO) 5 12.01 (d, J = 1.2 Hz, 1 H), 7.96 (dd, J = 11.5, 9.3 Hz, 1 H), 7.06 (dd, J = 12.1 , 6.9 Hz, 1 H), 2.61 (s, 3H).19F NMR (471 MHz, DMSO) 5 -126.32 (d, J = 23.7 Hz), -149.08 (d, J = 23.7 Hz). MS: The product was analysed by LCMS (Method 2): m / z does not ionise [M+H]+(ES+); 171.2 [M-H]' (ES-), at 1.20 min, >99% purity at 260nm + / - 80nm.1-(4,5-difluoro-2-hydroxy-3-nitrophenyl)ethan-1-one

[0082] Sulfuric acid (95%) (85.0 mL, 14.9 Eq, 1.56 mol) was added to 1-(4,5-difluoro-2- hydroxyphenyl)ethan-1-one (18.23 g, 99% Wt, 1 Eq, 104.8 mmol) and the solution was cooled to -5 °C (ice / brine bath). Nitric acid (69%) (11 g, 7.5 mL, 1.1 Eq, 0.12 mol) was added dropwise in portions over 40 min while maintaining the internal reaction temperature below 5 °C. The reaction mixture was stirred at 3 °C (ice / brine bath) for 30 min. An additional portion of nitric acid (69%) (1 g, 0.7 mL, 0.1 Eq, 0.01 mol) was added dropwise in portions while maintaining theinternal temperature below 5 °C and the reaction stirred for a further 10 min. The reaction mixture was poured onto crushed ice (~500 g), diluted with DCM (600 mL) and the stirred until the ice had melted and the solids had dissolved. The organic layer was collected and the aqueous was extracted with DCM (400 mL). The combined organic extracts were washed with brine (200 mL), dried over MgSO and concentrated in vacuo. The crude product was purified by chromatography on silica gel using a gradient of 0-10% 2-Me-THF / hexane to afford 1 -(4,5- difluoro-2-hydroxy-3-nitrophenyl)ethan-1-one (14.33 g, 62 mmol, 59 %) as an orange crystalline solid.

[0083] 1H NMR (500 MHz, DMSO) 5 8.43 (dd, J = 11 .0, 8.8 Hz, 1 H), 2.68 (s, 3H). (1 exchangeable proton not observed).19F NMR (471 MHz, DMSO) 5 -135.24 (d, J = 23.7 Hz), - 146.34 (d, J = 23.6 Hz). ). MS: The product was analysed by LCMS (Method 2): m / z 218.2 [M+H]+(ES+); 216.0 [M-H]’ (ES-), at 1.32 min, 94% purity at 260nm + / - 80nm.3-amino-4-fluoro-6-(1-iminoethyl)-2-nitrophenol

[0084] To a solution of 1-(4,5-difluoro-2-hydroxy-3-nitrophenyl)ethan-1-one (10.94 g, 94% Wt, 1 Eq, 47.36 mmol) in DMSO (95 mL) was added aq. Ammonia (30%) (23.9 mL, 7 Eq, 331.5 mmol). The reaction mixture was heated to 50 °C for 1 h and then allowed to cool to rt overnight. The reaction mixture was poured onto ice / water (~500 mL) and stirred for 30 min. The resultant precipitate mixture was cooled in an ice / water bath and the solids were collected by filtration, washing with water, to afford the desired product 3-amino-4-fluoro-6-(1-iminoethyl)- 2-nitrophenol as a yellow gum. The crude product was sucked dry for 10 min and then taken onto the next step without further purification assuming quantitative yield.

[0085] MS: The product was analysed by LCMS (Method 2): m / z 214.2 [M+H]+(ES+); 212.1 [M- H]’ (ES-), at 0.56 min, 95% purity 210-400nm.1-(4-amino-5-fluoro-2-hydroxy-3-nitrophenyl)ethan-1-one

[0086] To a suspension of 3-amino-4-fluoro-6-(1-iminoethyl)-2-nitrophenol (10.10 g, 1 Eq, 47.36 mmol) in MeCN (95 mL) was added aq. HCI (1 M) (49.73 mL, 1.05 Eq, 49.73 mmol) at rt. The reaction mixture was heated to 70 °C and stirred for 1 h. The resultant dark red solution was cooled to rt and EtOAc (800 mL) was added. The organic layer was washed with brine (2 x 200mL), dried over MgSO4and concentrated in vacuo to afford the desired product 1-(4-amino-5- fluoro-2-hydroxy-3-nitrophenyl)ethan-1-one (9.67 g, 45 mmol, 94 %) as a brown solid.

[0087] 1H NMR (500 MHz, DMSO) 5 14.22 (s, 1 H), 7.83 (d, J = 12.3 Hz, 1 H), 7.44 (s, 2H), 2.51 (s, 3H).19F NMR (471 MHz, DMSO) 5 -140.63. MS: The product was analysed by LCMS (Method 2): m / z 215.5 [M+H]+(ES+); 212.6 [M-H]’ (ES-), at 1.03 min, >99% purity 210-400nm. tert-butyl 4-(7-amino-6-fluoro-8-nitro-4-oxochroman-2-yl)piperidine-1-carboxylate

[0088] To a suspension of 1-(4-amino-5-fluoro-2-hydroxy-3-nitrophenyl)ethan-1-one (1.515 g, 99% Wt, 1 Eq, 7.0 mmol) and tert-butyl 4-formylpiperidine-1 -carboxylate (2.095 g, 1.403 Eq, 9.82 mmol) in EtOH (35 mL) at 50 °C was added pyrrolidine (630 pL, 1.10 Eq, 7.67 mmol) in one portion. The resultant solution was stirred at 50 °C for 6 h and then allowed to cool to rt. The reaction mixture was diluted with EtOAc (70 mL) and washed with brine (50 mL). The organic layer was collected and the aqueous was extracted with EtOAc (50 mL). The combined organic extracts were washed with brine (50 mL), dried over MgSO4and concentrated in vacuo to afford the crude product. The crude product was purified by chromatography on silica gel using a gradient of 0-5% MeOH / DCM to afford tert-butyl 4-(7-amino-6-fluoro-8-nitro-4- oxochroman-2-yl)piperidine-1 -carboxylate (3.08 g, 5.3 mmol, 75 %) as an orange foam.

[0089] MS: The product was analysed by UPLC (Method 1): m / z 310.5 [M+H-Boc]+(ES+);408.7 [M-H]’ (ES-), at 1.58 min, 70% purity 210-400nm tert-butyl 4-(7-amino-6-fluoro-8-nitro-4-oxo-4H-chromen-2-yl)piperidine-1 -carboxylate

[0090] A suspension of tert-butyl 4-(7-amino-6-fluoro-8-nitro-4-oxochroman-2-yl)piperidine-1- carboxylate (3.08 g, 70% Wt, 1 Eq, 5.27 mmol) and iodine (3.34 g, 2.5 Eq, 13.2 mmol) in pyridine (49 g, 50 mL, 120 Eq, 0.62 mol) was stirred at 100 °C for 4 h. Additional iodine (1.07 g, 0.8 Eq, 4.21 mmol) was added and stirred at 100 °C for 8 h and then allowed to cool to rt. The reaction mixture was concentrated in vacuo and the residue was azeotroped with toluene. The residue was taken up in DCM (200 mL) and washed with sat. aq. sodium thiosulfate (2 x 150 mL), sat. aq. NaHCO3(150 mL), brine (150 mL), dried over MgSO4and concentrated in vacuo to afford the crude product. The crude product was purified by chromatography on silica gel using a gradient of 0-5% MeOH / DCM to afford tert-butyl 4-(7-amino-6-fluoro-8-nitro-4-oxo- 4H-chromen-2-yl)piperidine-1 -carboxylate (882 mg, 1.8 mmol, 33 %) as a red oil.

[0091] MS: The product was analysed by UPLC (Method 1): m / z 307.8 [M+H-Boc]+(ES+);406.7 [M-H]- (ES-), at 1.47 min, 81 % purity 210-400nm7-amino-6-fluoro-8-nitro-2-(piperidin-4-yl)-4H-chromen-4-one di-hydrochloride

[0092] To a solution of tert-butyl 4-(7-amino-6-fluoro-8-nitro-4-oxo-4H-chromen-2-yl)piperidine-1 -carboxylate (882 mg, 81 % Wt, 1 Eq, 1.75 mmol) in DCM (20 mL) was added HCI (3 M in CPME) (5.0 mL, 8.6 Eq, 15 mmol) in one portion. The reaction was stirred at 40 °C for 2 h, additional HCI (3M in CPME) (2.0 mL, 3.4 Eq, 6.0 mmol) was added and the reaction heated for a further 3 h. The reaction mixture was cooled over an ice bath, the precipitate was collected by filtration, washed with DCM, and then sucked dry to afford 7-amino-6-fluoro-8-nitro-2-(piperidin- 4-yl)-4H-chromen-4-one di-hydrochloride (622 mg, 1.3 mmol, 76 %) as a brown solid.

[0093] 1H NMR (500 MHz, DMSO) 5 7.70 (d, J = 10.9 Hz, 1 H), 7.66 (s, 2H), 6.22 (s, 1 H), 3.36 (d, J = 12.6 Hz, 2H), 3.04 - 2.94 (m, 3H), 2.08 (d, J = 13.8 Hz, 2H), 1.92 - 1.75 (m, 2H).19F NMR (471 MHz, DMSO) 5 -129.93. MS: The product was analysed by UPLC (Method 1): m / z 308.5 [M+H]+(ES+) at 0.43 min, 82% purity 210-400nm2-(1-acetylpiperidin-4-yl)-7-amino-6-fluoro-8-nitro-4H-chromen-4-one

[0094] To a suspension of 7-amino-6-fluoro-8-nitro-2-(piperidin-4-yl)-4H-chromen-4-one di- hyddrochloride (622 mg, 82% Wt, 1 Eq, 1.34 mmol) and triethylamine (850 pL, 4.55 Eq, 6.10 mmol) in DCM (20 mL) was added acetyl chloride (150 pL, 1.57 Eq, 2.11 mmol) dropwise. The reaction mixture was stirred at rt for 2 h. Additional acetyl chloride (35.0 pL, 0.367 Eq, 492 pmol) and triethylamine (220 pL, 1.18 Eq, 1.58 mmol) were added and stirred for 1 h. The reaction mixture was concentrated in vacuo and the residue was partitioned between DCM (150 mL) and sat. aq. NaHCO3(100mL). The organic layer was collected and the aqueous was extracted with DCM (2 x 50 mL). The combined organic extracts were washed with brine (100 mL), dried over MgSO and concentrated in vacuo to afford the crude product. The crude product was purified by chromatography on silica gel using a gradient of 0-10% MeOH / DCM to afford 2-(1- acetylpiperidin-4-yl)-7-amino-6-fluoro-8-nitro-4H-chromen-4-one (336 mg, 0.93 mmol, 70 %) as a yellow solid.

[0095] 1H NMR (500 MHz, DMSO) 5 7.69 (d, J = 11 .0 Hz, 1 H), 7.64 (s, 2H), 6.20 (s, 1 H), 4.47 (d, J = 13.1 Hz, 1 H), 3.91 (d, J = 13.6 Hz, 1 H), 3.19 - 3.08 (m, 1 H), 2.87 (ddt, J = 11.7, 7.1 , 3.7Hz, 1 H), 2.66 - 2.57 (m, 1 H), 2.02 (s, 3H), 1.99 - 1.87 (m, 2H), 1.59 (qd, J = 12.3, 4.2 Hz, 1 H), 1.45 (qd, J = 12.5, 4.4 Hz, 1 H).19F NMR (471 MHz, DMSO) 5 -130.11 . MS: The product was analysed by UPLC (Method 1): m / z 350.5 [M+H]+(ES+); 348.5 [M-H]' (ES-), at 0.89 min, 97% purity 210-400nm8-(1-acetylpiperidin-4-yl)-4-fluoro-2-(trifluoromethyl)chromeno[7,8-d1imidazol-6(3H)-one

[0096] To a solution of 2-(1-acetylpiperidin-4-yl)-7-amino-6-fluoro-8-nitro-4H-chromen-4-one (336 mg, 97% Wt, 1 Eq, 933 pmol) and triethylamine (1.85 mL, 14.2 Eq, 13.3 mmol) in THF (14.8 mL), EtOH (14.8 mL) and Water (1.48 mL) was added sodium dithionite (248 pL, 3.82 Eq, 3.56 mmol). The reaction mixture was stirred at 60 °C for 1 h and then concentrated in vacuo. The crude intermediate was suspended in TFA (4 mL, 60 Eq, 0.05 mol) and stirred at 70 °C for 8 h. The reaction was allowed to cool to rt, concentrated in vacuo and the residue was azeotroped with DCM (3 x 10 mL) to afford the crude product. The crude product was dried onto Celite and purified by chromatography on silica gel using a gradient of 0-10% (0.7 M Ammonia in MeOH) / DCM to afford 8-(1-acetylpiperidin-4-yl)-4-fluoro-2-(trifluoromethyl)chromeno[7,8- d]imidazol-6(3H)-one (428 mg, 0.62 mmol, 67 %) as an orange gum.

[0097] 1H NMR (500 MHz, MeOD) 5 7.70 (d, J = 10.1 Hz, 1 H), 6.37 (s, 1 H), 4.72 (dt, J = 13.4, 2.3 Hz, 1 H), 4.14 - 4.07 (m, 1 H), 3.09 (tt, J = 11.9, 3.6 Hz, 1 H), 2.79 (td, J = 13.0, 2.8 Hz, 1 H), 2.22 - 2.07 (m, 5H), 1.94 (qd, J = 12.6, 4.3 Hz, 1 H), 1.82 (qd, J = 12.6, 4.3 Hz, 1 H).19F NMR (471 MHz, MeOD) 5 -65.62, -131.83. MS: The product was analysed by UPLC (Method 1): 3943-234-1 , m / z 398.1 [M+H]+(ES+); 396.2 [M-H]' (ES-), at 1.06 min, >99% purity 210-400nm8-(1-acetylpiperidin-4-yl)-3-ethyl-4-fluoro-2-(trifluoromethyl)chromeno[7,8-d1imidazol-6(3H)-one(1) & 8-(1-acetylpiperidin-4-yl)-1-ethyl-4-fluoro-2-(trifluoromethyl)chromeno[7,8-d1imidazol-6(1 H)-one (2)

[0098] A solution of 8-(1-acetylpiperidin-4-yl)-4-fluoro-2-(trifluoromethyl)chromeno[7,8- d]imidazol-6(3H)-one (200 mg, 58% Wt, 1 Eq, 292 pmol), iodoethane (77.0 pL, 3.27 Eq, 956 pmol) and DIPEA (167 mg, 225 pL, 4.42 Eq, 1.29 mmol) in DMF (3 mL) was stirred at 65 °C for 2 h. The reaction mixture was diluted with water (10 mL) and then extracted with DCM (2 x 5 mL). The combined organic extracts were dried over a phase separator and concentrated in vacuo. The residue was azeotroped with toluene to afford the crude product. The crude product was purified by chromatography on silica gel using a gradient of 0-10% MeOH / DCM to afford a mixture of regioisomers. The mixture (137 mg) was dissolved in 2.1 mL with DMSO, filtered and purified by reversed phase preparative HPLC (Waters 2767 Sample Manager, Waters 2545 Binary Gradient Module, Waters Systems Fluidics Organiser, Waters 515 ACD pump, Waters 515 Makeup pump, Waters 2998 Photodiode Array Detector, Waters QDa) on a Waters X- Select CSH C18 ODB prep column, 130A, 5 pm, 30 mm X 100 mm, flow rate 40 mL min'1eluting with a 0.1 % Formic acid in water-MeCN gradient over 17.5 mins using UV across all wavelengths with PDA as well as a QDA and ELS detector. The clean fractions were evaporated in a Genevac to afford:Compound 1

[0099] 8-(1-acetylpiperidin-4-yl)-3-ethyl-4-fluoro-2-(trifluoromethyl)chromeno[7,8-d]imidazol- 6(3H)-one (12.0 mg, 28 pmol, 9.6 %) as a white solid.

[0100] 1H NMR (500 MHz, MeOD) 5 7.80 (d, J = 11.3 Hz, 1 H), 6.39 (s, 1 H), 4.72 (d, J = 13.4 Hz, 1 H), 4.65 (q, J = 7.2 Hz, 2H), 4.11 (d, J = 13.9 Hz, 1 H), 3.34 - 3.26 (m, 1 H), 3.14 - 3.05 (m, 1 H), 2.84 - 2.75 (m, 1 H), 2.17 (s, 5H), 1.93 (qd, J = 12.3, 4.1 Hz, 1 H), 1.80 (qd, J = 12.5, 4.3 Hz, 1 H), 1.58 (t, J = 7.2 Hz, 3H).19F NMR (471 MHz, MeOD) 5 -63.70, -136.08. MS: The product was analysed by LCMS (Method 2): m / z 426.0 [M+H]+(ES+) 424.0 [M-H]' (ES-) at 1.34 min, 99% purity at 260nm + / - 80nm.Compound 2

[0101] 8-(1-acetylpiperidin-4-yl)-1-ethyl-4-fluoro-2-(trifluoromethyl)chromeno[7,8-d]imidazol- 6(1 H)-one (38.0 mg, 88 pmol, 30 %) as a white solid.

[0102] 1H NMR (500 MHz, MeOD) 6 7.69 (d, J = 10.0 Hz, 1 H), 6.43 (s, 1 H), 4.81 (q, J = 7.2 Hz, 2H), 4.75 - 4.69 (m, 1 H), 4.15 - 4.09 (m, 1 H), 3.35 - 3.27 (m, 1 H), 3.13 (tt, J = 12.0, 3.6 Hz, 1 H), 2.84 - 2.76 (m, 1 H), 2.22 - 2.11 (m, 5H), 1.84 (qd, J = 12.7, 4.4 Hz, 1 H), 1.74 (qd, J = 12.6, 4.4 Hz, 1 H), 1.64 (t, J = 7.2 Hz, 3H).19F NMR (471 MHz, MeOD) 5 -63.71 , -131.09. MS: The product was analysed by LCMS (Method 2): m / z 426.0 [M+H]+(ES+); 424.0 [M-H]’ (ES-), at 1 .29 min, 99% purity at 260nm + / - 80nm.Compounds 3 & 44-fluoro-8-(piperidin-4-yl)-2-(trifluoromethyl)chromenof7,8-d1imidazol-6(3H)-one trifluoroacetate

[0103] To a solution of tert-butyl 4-(7-amino-6-fluoro-8-nitro-4-oxo-4H-chromen-2-yl)piperidine- 1 -carboxylate (2.32 g, 76% Wt, 1 Eq, 4.33 mmol) and triethylamine (12.0 mL, 19.9 Eq, 86.1 mmol) in THF (40.0 mL), EtOH (40.0 mL) and Water (4.00 mL) was added sodium dithionite (1.61 mL, 5.34 Eq, 23.1 mmol). The reaction mixture was stirred at 60 °C for 1 h and then concentrated in vacuo. The residue was azeotroped with DCM to afford the crude product as a mixture of tert-butyl 4-(7,8-diamino-6-fluoro-4-oxo-4H-chromen-2-yl)piperidine-1 -carboxylate and (7-amino-2-(1-(tert-butoxycarbonyl)piperidin-4-yl)-6-fluoro-4-oxo-4H-chromen-8-yl)sulfamic acid. To the crude mixture was added TFA (10.0 mL, 30.0 Eq, 130 mmol). The reaction mixture was stirred at 70 °C for 3 h and then concentrated in vacuo. The residue was azeotroped with DCM to afford the crude product 4-fluoro-8-(piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8- d]imidazol-6(3H)-one as the trifluoroacetic acid salt in quantitative yield, as a red oil. The crude product was used without further purification.

[0104] MS: The product was analysed by LCMS (Method 3): m / z 356.0 [M+H]+(ES+); 354.0 [M- H]’ (ES-), at 0.67 min, 63% purity at 260nm + / - 90nm. tert-butyl 4-(4-fluoro-6-oxo-2-(trifluoromethyl)-3,6-dihvdrochromenof7,8-d1imidazol-8- yl)piperidine-1 -carboxylate

[0105] To a suspension of crude 4-fluoro-8-(piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8- d]imidazol-6(3H)-one TFA (1.54 g, 100% Wt, 1 Eq, 4.33 mmol) in THF (50.0 mL) was added sodium carbonate (10.3 g, 22.4 Eq, 97.2 mmol). The mixture was stirred for 5 min. at rt and then Boc-anhydride (1.49 mL, 1.50 Eq, 6.51 mmol) was added in one portion. The reaction mixture was stirred at rt for 1 h. The reaction mixture was diluted with EtOAc (100 mL) and filtered, washing with EtOAc. The filtrate was concentrated in vacuo to afford the crude product. The crude product was purified by chromatography on silica gel using a gradient of 10% (3:1 EtOAc: EtOH) / DCM to afford tert-butyl 4-(4-fluoro-6-oxo-2-(trifluoromethyl)-3,6- dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1-carboxylate (700 mg, 1.5 mmol, 34 %) as an orange solid.

[0106] 1H NMR (500 MHz, MeOD) 5 7.70 (d, J = 10.1 Hz, 1 H), 6.36 (s, 1 H), 4.28 (d, J = 13.4 Hz, 2H), 3.04 - 2.86 (m, 3H), 2.11 (d, J = 13.0 Hz, 2H), 1.81 (qd, J = 12.5, 4.3 Hz, 2H), 1.49 (s, 9H). 1 proton not observed in MeOD.19F NMR (471 MHz, MeOD) 5 -65.63, -131.87. MS: The product was analysed by LCMS (Method 3): m / z 478.0 (M+Na)+ (ES+); 454.0 [M-H]' (ES-), at 0.94 min, 96% purity at 260nm + / - 90nm. tert-butyl 4-(3-ethyl-4-fluoro-6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d1imidazol-8- yl)piperidine-1 -carboxylate & tert-butyl 4-(1-ethyl-4-fluoro-6-oxo-2-(trifluoromethyl)-1 ,6- dihvdrochromeno[7,8-d1imidazol-8-yl)piperidine-1-carboxylate

[0107] A solution of tert-butyl 4-(4-fluoro-6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8- d]imidazol-8-yl)piperidine-1 -carboxylate (288 mg, 96% Wt, 1 Eq, 607 pmol), DI PEA (450 pL, 4.26 Eq, 2.58 mmol) and ethyl iodide (150 pL, 3.06 Eq, 1.86 mmol) in DMF (3 mL) was stirred at 65 °C for 3 h. The reaction mixture was diluted with EtOAc (50 mL) and washed with water (25 mL), brine (2 x 25 mL), dried over MgSO and concentrated in vacuo to afford the crude product. The crude product was purified by chromatography on silica gel using a gradient of 0- 5% MeOH / DCM to afford a mixture of tert-butyl 4-(3-ethyl-4-fluoro-6-oxo-2-(trifluoromethyl)-3,6- dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1-carboxylate and tert-butyl 4-(1-ethyl-4-fluoro- 6-oxo-2-(trifluoromethyl)-1 ,6-dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1 -carboxylate (296 mg, 0.57 mmol, 94 %) as an orange solid.

[0108] MS: The product was analysed by LCMS (Method 3): m / z 506.2 (M+Na)+ (ES+); 482.0 [M-H]' (ES-), at 1.78 / 1.83 min, 30 / 63% purity at 260nm + / - 90nm.3-ethyl-4-fluoro-8-(piperidin-4-yl)-2-(trifluoromethyl)chromenof7,8-d1imidazol-6(3H)-one hydrochloride & -ethyl-4-fluoro-8-(piperidin-4-yl)-2-(trifluoromethyl)chromenof7,8-d1imidazol- 6(1 H)-one hydrochloride

[0109] To a solution of tert-butyl 4-(3-ethyl-4-fluoro-6-oxo-2-(trifluoromethyl)-3,6- dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1-carboxylate and tert-butyl 4-(1-ethyl-4-fluoro- 6-oxo-2-(trifluoromethyl)-1 ,6-dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1 -carboxylate (296 mg, 93% Wt, 1 Eq, 569 pmol) in DCM (3.00 mL) was added HCI (3 M in CPME) (219 mg, 2.00 mL, 3.00 molar, 10.5 Eq, 6.00 mmol). The reaction mixture was stirred at rt for 18 h and then concentrated in vacuo. The residue was azeotroped with DCM to afford the crude product as a mixture of 3-ethyl-4-fluoro-8-(piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol-6(3H)- one hydrochloride and 1-ethyl-4-fluoro-8-(piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8- d]imidazol-6(1 H)-one hydrochloride (239 mg, 0.54 mmol, 95 %) as a pink solid.

[0110] MS: The product was analysed by LCMS (Method 3): m / z 384.2 [M+H]+(ES+); 382.0 [M- H]’ (ES-), at 1.35 / 1.43 min, 31 / 64% purity at 260nm + / - 90nm.3-ethyl-4-fluoro-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromenof7,8-d1imidazol-6(3H)-one (3) & 1-ethyl-4-fluoro-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromenof7,8-d1imidazol- 6(1H)-one (4)

[0111] To a suspension of crude 3-ethyl-4-fluoro-8-(piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol-6(3H)-one hydrochloride and 1-ethyl-4-fluoro-8-(piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol-6(1 H)-one hydrochloride (251 mg,95% Wt, 1.00 Eq, 0.569 mmol) in THF (5 mL) was added formaldehyde (37 wt% in water) (220 pL, 37% Wt, 5.19 Eq, 2.95 mmol). The mixture was stirred at rt for 1 h and then sodium triacetoxyborohydride (217 mg, 1.80 Eq, 1.02 mmol) was added in one portion. The reaction mixture was stirred at rt for 2 h. Additional sodium triacetoxyborohydride (100 mg, 0.829 Eq, 472 pmol) was added and stirred for 1 h. The reaction mixture was quenched with MeOH (~5 mL) and then concentrated in vacuo to afford the crude product. The crude product was purified by chromatography on silica gel using a gradient of 0-5% (0.7 M Ammonia in MeOH) / DCM to afford a mixture of regioisomers. The mixture (90.0 mg) was dissolved to 11.3 mg / mL in MeOH with sonication, filtered and was then separated by chiral SFC on a Sepiatec with UV detection at 220 nm, 40 °C, 100 bar. The column was a ChiralpaK IH, 10 x 250mm, 5pm, flow rate 20mL / min at 15% MeOH (0.2% ammonia), 85% CO2. The clean fractions were pooled, rinsed with methanol, and concentrated to dryness using a rotary evaporator. The residues were redissolved in methanol, transferred into final vials and evaporated on a Biotage V10. The samples were then further dried in a vacuum oven at 30 °C / 5mbar overnight to afford:Compound 3

[0112] 3-ethyl-4-fluoro-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol- 6(3H)-one (48.9 mg, 0.12 mmol, 21 %) as an orange powder.

[0113] 1H NMR (500 MHz, DMSO) 5 7.72 (d, J = 11.2 Hz, 1 H), 6.34 (s, 1 H), 4.54 (q, J = 7.2 Hz, 2H), 2.92 - 2.85 (m, 2H), 2.70 (ddt, J = 12.0, 8.2, 3.7 Hz, 1 H), 2.20 (s, 3H), 2.04 - 1.92 (m, 4H), 1.75 (qd, J = 12.6, 4.0 Hz, 2H), 1.48 (t, J = 7.1 Hz, 3H).19F NMR (471 MHz, DMSO) 5 -61.27, - 134.50. MS: The product was analysed by LCMS (Method 3): m / z 398.1 [M+H]+(ES+); no ionisation (ES-), at 1.41 min, 99% purity 210-400nm.Compound 4

[0114] 1-ethyl-4-fluoro-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol- 6(1 H)-one (106 mg, 0.26 mmol, 46 %) as a yellow solid.

[0115] 1H NMR (500 MHz, DMSO) 5 7.61 (d, J = 10.1 Hz, 1 H), 6.41 (s, 1 H), 4.72 (q, J = 7.1 Hz, 2H), 2.90 (d, J = 11 .6 Hz, 2H), 2.72 (tt, J = 11 .9, 3.7 Hz, 1 H), 2.21 (s, 3H), 2.03 - 1 .90 (m, 4H), 1.78 (qd, J = 12.5, 4.0 Hz, 2H), 1.55 (t, J = 7.1 Hz, 3H).19F NMR (471 MHz, DMSO) 5 -61.34, -129.99. MS: The product was analysed by LCMS (Method 3): m / z 398.2 [M+H]+(ES+); 396.2 [M-H]' (ES-), at 1.42 min, >99% purity at 260nm + / - 90nm.Compound 5 & 6 tert-butyl 4-(4-fluoro-3-methyl-6-oxo-2-(trifluoromethyl)-3,6-dihydrochromenoR,8-d1imidazol-8- yl)piperidine-1 -carboxylate & tert-butyl 4-(4-fluoro-1-methyl-6-oxo-2-(trifluoromethyl)-1 ,6- dihydrochromenoR,8-d1imidazol-8-yl)piperidine-1-carboxylate

[0116] A solution of tert-butyl 4-(4-fluoro-6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8- d]imidazol-8-yl)piperidine-1-carboxylate (292 mg, 96% Wt, 1 Eq, 616 pmol), DIPEA (450 pL, 4.20 Eq, 2.58 mmol) and lodomethane (110 pL, 2.86 Eq, 1.76 mmol) in DMF (3 mL) was stirred at 65 °C for 3 h. The reaction mixture was diluted with EtOAc (50 mL) and washed with water (25 mL), brine (2 x 25 mL), dried over MgSO and concentrated in vacuo to afford the crude product. The crude product was purified by chromatography on silica gel using a gradient of 0- 5% MeOH / DCM to afford a mixture of tert-butyl 4-(4-fluoro-3-methyl-6-oxo-2-(trifluoromethyl)- 3,6-dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1-carboxylate and tert-butyl 4-(4-fluoro-1- methyl-6-oxo-2-(trifluoromethyl)-1 ,6-dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1- carboxylate (286 mg, 0.60 mmol, 97 %) as an orange solid.

[0117] MS: The product was analysed by LCMS (Method 3): m / z 492.0 [M+Na]+(ES+); 468.0 [M-H]' (ES-), at 1.71 / 1.73 min, 33 / 65% purity at 260nm + / - 90nm.4-fluoro-3-methyl-8-(piperidin-4-yl)-2-(trifluoromethyl)chromenoR,8-d1imidazol-6(3H)-one hydrochloride & 4-fluoro-1-methyl-8-(piperidin-4-yl)-2-(trifluoromethyl)chromenoR,8-d1imidazol- 6(1 H)-one hydrochloride

[0118] To a solution of crude tert-butyl 4-(4-fluoro-3-methyl-6-oxo-2-(trifluoromethyl)-3,6- dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1-carboxylate and tert-butyl 4-(4-fluoro-1- methyl-6-oxo-2-(trifluoromethyl)-1 ,6-dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1- carboxylate (286 mg, 98% Wt, 1 Eq, 597 pmol) in DCM (3.00 mL) was added HCI (3 M in CPME) (2.00 mL, 10.0 Eq, 6.00 mmol). The reaction mixture was stirred at rt for 18 h and then concentrated in vacuo. The residue was azeotroped with DCM to afford the crude product as a mixture of 4-fluoro-3-methyl-8-(piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol-6(3H)-one hydrochloride and 4-fluoro-1-methyl-8-(piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8- d]imidazol-6(1 H)-one hydrochloride (242 mg, 596 pmol, 99 %) as a pink solid.

[0119] MS: The product was analysed by LCMS (Method 3): m / z 370.0 [M+H]+(ES+); 368.2 [M- H]’ (ES-), at 1 .25 / 1 .29 min, 29 / 70% purity at 260nm + / - 90nm.4-fluoro-3-methyl-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d1imidazol-6(3H)- one (5) & 4-fluoro-1-methyl-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromenor7,8- d1imidazol-6(1 H)-one (6)

[0120] To a suspension of crude 4-fluoro-3-methyl-8-(piperidin-4-yl)-2- (trifluoromethyl)chromeno[7,8-d]imidazol-6(3H)-one hydrochloride and 4-fluoro-1-methyl-8- (piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol-6(1 H)-one hydrochloride (242 mg, 100% Wt, 1.00 Eq, 0.597 mmol) in THF (5.00 mL) was added formaldehyde (37 wt% in water) (225 pL, 5.06 Eq, 3.02 mmol). The mixture was stirred at rt for 1 h and then sodium triacetoxyborohydride (217 mg, 1.72 Eq, 1.02 mmol) was added in one portion. The reaction mixture was stirred at rt for 2 h. Additional sodium triacetoxyborohydride (100 mg, 0.790 Eq, 472 pmol) was added and stirred for 1 h. The reaction mixture was quenched with MeOH (~5 mL) and then concentrated in vacuo to afford the crude product. The crude product was purified by chromatography on silica gel using a gradient of 0-5% (0.7 M Ammonia in MeOH) / DCM to afford a mixture of isomers as a pink solid. The mixture (203 mg) was dissolved to 20.3 mg / mL in 10 mL MeOH with sonication, filtered and was then separated by chiral SFC on a Waters Prep 100 with a PDA and QDa detectors, 40 °C, 120 bar. The column was a Phenomenex C3, 20 x 250mm, 5pm, flow rate 65mL / min at 10% MeOH (0.3% ammonia), 90% CO2. The clean fractions were pooled, rinsed with methanol, and concentrated to dryness using a rotaryevaporator. The residues were re-dissolved in methanol, transferred into final vials and evaporated on a Biotage V10 to afford:Compound 5

[0121] 4-fluoro-3-methyl-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol- 6(3H)-one (48.9 mg, 128 pmol, 21.4 %) as an orange-pink solid.

[0122] 1H NMR (500 MHz, DMSO) 6 7.69 (d, J = 11.4 Hz, 1 H), 6.33 (s, 1 H), 4.16 (s, 3H), 2.88 (d, J = 11.7 Hz, 2H), 2.69 (tt, J = 11.8, 3.7 Hz, 1 H), 2.20 (s, 3H), 1.98 (qd, J = 11.2, 2.8 Hz, 4H),I .75 (qd, J = 12.5, 3.8 Hz, 2H).19F NMR (471 MHz, DMSO) 5 -61.49, -134.17. MS: The product was analysed by LCMS (Method 2): m / z 384.5 [M+H]+(ES+); no ionisation (ES-), at 0.71 min, >99% purity 210-400nm.Compound 6

[0123] 4-fluoro-1-methyl-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol- 6(1 H)-one (137.5 mg, 0.34 mmol, 58 %) as an orange-pink solid.

[0124] 1H NMR (500 MHz, DMSO) 5 7.60 (d, J = 10.1 Hz, 1 H), 6.39 (s, 1 H), 4.33 (d, J = 1.2 Hz, 3H), 2.89 (dt, J = 11.9, 3.3 Hz, 2H), 2.72 (tt, J = 11.9, 3.7 Hz, 1 H), 2.20 (s, 3H), 1.97 (qd, J =I I .0, 3.0 Hz, 4H), 1 .78 (qd, J = 12.4, 3.8 Hz, 2H).19F NMR (471 MHz, DMSO) 5 -61.53, - 130.05. MS: The product was analysed by LCMS (Method 2): m / z 384.5 [M+H]+(ES+); no ionisation (ES-), at 0.69 min, 98% purity 210-400nm.Compounds 7 & 8 tert-butyl 4-(4-fluoro-3-(methyl-d3)-6-oxo-2-(trifluoromethyl)-3,6-dihydrochromenoR,8- d1imidazol-8-yl)piperidine-1 -carboxylate & tert-butyl 4-(4-fluoro-1-(methyl-d3)-6-oxo-2- (trifluoromethyl)-1 ,6-dihvdrochromeno(7,8-d1imidazol-8-yl)piperidine-1 -carboxylate

[0125] A stirred solution of tert-butyl 4-(4-fluoro-6-oxo-2-(trifluoromethyl)-3,6- dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1-carboxylate (200 mg, 94% Wt, 1 Eq, 413 pmol), DIPEA (310 pL, 4.31 Eq, 1.78 mmol) and iodomethane-d3 (70.0 pL, 2.72 Eq, 1.12 mmol) in THF (2.50 mL) was heated at 65 °C for 1 h. The reaction mixture was concentrated in vacuoto afford the crude product. The crude product was purified by column chromatography on silica gel using a gradient of 0-30% (3:1 , EtOAc / EtOH) / DCM to afford a mixture of tert-butyl 4-(4- fluoro-3-(methyl-d3)-6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8-d]imidazol-8- yl)piperidine-1 -carboxylate and tert-butyl 4-(4-fluoro-1-(methyl-d3)-6-oxo-2-(trifluoromethyl)-1 ,6- dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1-carboxylate (192 mg, 0.40 mmol, 97 %) in quantitative yields, as a pale orange solid.

[0126] MS: The product was analysed by LCMS (Method 3): m / z 495.0 [M+Na]+(ES+); 471.1 [M-H]' (ES-), at 1.66 / 1.68 min, 99% purity at 260nm + / - 90nm.4-fluoro-3-(methyl-d3)-8-(piperidin-4-yl)-2-(trifluoromethyl)chromenoR,8-d1imidazol-6(3H)-one hydrochloride & 4-fluoro-1-(methyl-d3)-8-(piperidin-4-yl)-2-(trifluoromethyl)chromenoR,8- d1imidazol-6(1 H)-one hydrochloride

[0127] To a stirred solution of tert-butyl 4-(4-fluoro-3-(methyl-d3)-6-oxo-2-(trifluoromethyl)-3,6- dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1-carboxylate and tert-butyl 4-(4-fluoro-1- (methyl-d3)-6-oxo-2-(trifluoromethyl)-1 ,6-dihydrochromeno[7,8-d]imidazol-8-yl)piperidine-1- carboxylate (192 mg, 99% Wt, 1 Eq, 402 pmol) in DCM (2.00 mL) was added HCI (3 M solution in CPME) (1.35 mL, 10.1 Eq, 4.05 mmol). The resultant suspension was stirred at rt for 18 h and then concentrated in vacuo to afford the crude product as a mixture of 4-fluoro-3-(methyl-d3)-8- (piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol-6(3H)-one hydrochloride and 4- fluoro-1-(methyl-d3)-8-(piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol-6(1 H)-one hydrochloride (187 mg, 0.40 mmol) in quantitative yield, as a pale pink solid.

[0128] MS: The product was analysed by LCMS (Method 3): m / z 373.0 [M+H]+(ES+); 371.0 [M- H]’ (ES-), at 1 .25 / 1 .30 min, 98% purity at 260nm + / - 90nm.4-fluoro-3-(methyl-d3)-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromenoR,8-d1imidazol-6(3H)-one (7) & 4-fluoro-1-(methyl-d3)-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromenoR,8-d1imidazol-6(1 H)-one (8)

[0129] A suspension of 4-fluoro-1-(methyl-d3)-8-(piperidin-4-yl)-2- (trifluoromethyl)chromeno[7,8-d]imidazol-6(1 H)-one hydrochloride and 4-fluoro-3-(methyl-d3)-8- (piperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8-d]imidazol-6(3H)-one hydrochloride (184 mg, 98% Wt, 1.00 Eq, 396 pmol) and formaldehyde (37 wt% in water) (150 pL, 5.09 Eq, 2.01 mmol) in THF (5.00 mL) was stirred at rt for 1 h. Sodium triacetoxyborohydride (225 mg, 2.68 Eq, 1.06 mmol) was added in one portion and the reaction mixture was stirred at rt for 30 min. The reaction was quenched with MeOH (5 mL) and concentrated in vacuo to afford the crude product. The crude product was purified by column chromatography on silica gel using a gradient of 0-6% (0.7 M Ammonia in MeOH) / DCM to afford a mixture of products as a pale orange solid. The mixture (135 mg) was dissolved in a suitable mixture of mobile phases and DMSO to ensure compete solvation then filtered and purified by preparative HPLC (Waters 2767 Sample Manager, Waters 2545 Binary Gradient Module, Waters Systems Fluidics Organiser, Waters 515 ACD pump, Waters 515 Makeup pump, Waters 2998 Photodiode Array Detector, Waters QDa) on a Waters XBridge BEH C18 ODB prep column, 130A, 5 pm, 30 mm X 100 mm, flow rate 40 mL min-1 eluting with a 0.3% Ammonia in water-MeCN gradient over 12.5 mins using UV across all wavelengths with PDA as well as a QDA and ELS detector. The clean fractions were evaporated in a Genevac to afford a mixture of regioisomers. The mixture (82 mg) was dissolved to 15 mg / mL in MeOH with sonication, filtered and then separated by chiral SFC on a Waters Prep 15 with a PDA detector, 40 °C, 120 bar. The column was a ChiralpaK IH, 10 x 250mm, 5pm, flow rate 15 mL / min at 15% MeOH (0.4% DEA), 85% CO2. The clean fractions were pooled, rinsed with MeOH, and concentrated to dryness using a rotary evaporator. The residues were re-dissolved in MeOH, transferred into final vials and evaporated on a Biotage V10 to afford:Compound 7

[0130] 4-fluoro-3-(methyl-d3)-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8- d]imidazol-6(3H)-one (17.7 mg, 45 pmol, 11 %) as a white solid.

[0131] 1H NMR (500 MHz, DMSO) 5 7.69 (d, J = 11 .4 Hz, 1 H), 6.33 (s, 1 H), 2.88 (dt, J = 11 .9, 3.3 Hz, 2H), 2.69 (tt, J = 11.8, 3.6 Hz, 1 H), 2.20 (s, 3H), 2.04 - 1.93 (m, 4H), 1.75 (qd, J = 12.5, 3.8 Hz, 2H).19F NMR (471 MHz, DMSO) 5 -61.51 , -134.21. MS: The product was analysed by UPLC (Method 2): m / z 387.0 [M+H]+(ES+); no ionisation (ES-), at 1.28 min, 99% purity 210- 400nm.Compound 8

[0132] 4-fluoro-1-(methyl-d3)-8-(1-methylpiperidin-4-yl)-2-(trifluoromethyl)chromeno[7,8- d]imidazol-6(1 H)-one (57.1 mg, 0.15 mmol, 37 %) as a white solid.

[0133] 1H NMR (500 MHz, DMSO) 5 7.59 (d, J = 10.1 Hz, 1 H), 6.39 (s, 1 H), 2.89 (dt, J = 11.9, 3.3 Hz, 2H), 2.72 (tt, J = 11.9, 3.6 Hz, 1 H), 2.20 (s, 3H), 2.03 - 1.92 (m, 4H), 1.78 (qd, J = 12.3, 3.8 Hz, 2H).19F NMR (471 MHz, DMSO) 5 -61.56, -130.05. MS: The product was analysed by UPLC (Method 2): m / z 387.0 [M+H]+(ES+); no ionisation (ES-), at 1.26 min, 99% purity 210- 400nm.EXAMPLE 2: EFFECTS OF TEST COMPOUNDS ON SURVIVAL OF MOTOR NEURONS, INTEGRITY OF THE NEURITE NETWORK AND TDP43 MISLOCALISATION IN PRIMARY SOD1 TRANSGENIC SPINAL CORD MOTOR NEURONS FOLLOWING GLUTAMATE INSULT

[0134] The aim of this study was to assess the neuroprotective effects of novel compounds (at several concentrations) in an in vitro model of ALS, based on a primary culture of spinal motor neurons, from SOD1 G93A rat embryos, injured with glutamate. Survival of motor neurons, integrity of the neurite network and TDP43 mislocalisation were evaluated.Methods:Primary culture of spinal motor neurons

[0135] Rat spinal cord motor neurons (MNs) were cultured as described by Boussicault et al., 2020 and Wang et al., 2013. Briefly, pregnant female rats of 14 days gestation were killed using a deep anesthesia with CO2chamber and a cervical dislocation. Then, embryos were removed from the uterus and immediately placed in ice-cold L15 Leibovitz medium with a 2 % penicillin(10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1 % bovine serum albumin (BSA). Only Tg SOD1G93A embryos were used to prepare the spinal MN culture

[0136] The whole spinal cord was extracted from each embryo and spinal cords from transgenic SOD1G93A embryos were pooled in ice-cold medium of Leibovitz (L15). Spinal cords were then treated for 20 min at 37 °C with a trypsin-EDTA solution at a final concentration of 0.05 % trypsin and 0.02 % Ethylenediaminetetraacetic acid (EDTA). The dissociation was stopped by addition of Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g / L of glucose, containing DNAse I grade II (final concentration 0.5 mg / ml) and 10 % fetal bovine serum (FBS). Cells were mechanically dissociated by three forced passages through the tip of a 10-mL pipette. Cells were then centrifuged at 515 x g for 10 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in a defined culture medium consisting of Neurobasal medium with a 2 % solution of B27 supplement, 2 mM of L-glutamine, 2 % of PS solution, and 10 ng / mL of brain- derived neurotrophic factor (BDNF). Viable cells were counted in a Neubauer cytometer, using the trypan blue exclusion test. The cells were seeded at a density of 20,000 per well in 96-well plates precoated with poly-L-lysine and were cultured at 37 °C in an air (95 %)-CO2 (5 %) incubator. The medium was changed every other day. Only 60 wells of the 96-well plates were used for the culture: the wells of first and last lines and columns were not used and filled with sterile water to avoid any edge effect.Test compounds and glutamate exposure

[0137] Vehicle: Culture medium (0.1 % dimethyl sulfoxide (DMSO) as final concentration)

[0138] Pre-incubation: On day 13 of culture, test compounds were solubilized in DMSO, then diluted in the culture medium and pre-incubated with primary neurons for 1 h, before glutamate injury. BDNF, solubilized in PBS, was diluted in the culture medium and pre-incubated with primary neurons for 1 h, before glutamate insult. BDNF, was used as a validated positive experimental control.

[0139] Glutamate injury: On day 13 of culture, half of the culture medium (100 pL) was removed from each well and replaced by culture medium containing 10 pM glutamate (so that final concentration of glutamate is 5 pM, except for control condition) and the compounds (2-fold concentrated) for 20 min. After 20 min, glutamate was washed out and fresh culture medium with the compounds or vehicle was added for an additional 24 h period.ORGANISATION OF CULTURE PLATES

[0140] Compounds were tested in 96-well plates (n = 6 culture wells per condition).

[0141] Culture medium was adjusted to 0.1 % DMSO for all the experimental conditions. The stock solution of the test compounds was made at 10 mM in pure DMSO, aliquoted and stored at -80 °C. Next, compounds were submitted to a serial dilution to obtain 1000-fold concentrated stock solutions in DMSO.END POINT EVALUATIONImmunostaininq: MAP-2 and TDP43

[0142] 24 hours after glutamate application, the supernatants were collected and stored at -80 °C, and cells were fixed by a cold solution of ethanol (95 %) and acetic acid (5 %) for 5 min at - 20 °C. Cell membranes were permeabilized and non-specific binding sites was blocked with a solution of PBS containing 0.1 % of saponin and 1 % FBS for 15 min at room temperature. Cells were incubated for 2 h with the following primary antibodies: a) a mouse monoclonal antibody anti microtubule-associated-protein 2 (MAP-2) at dilution of 1 / 400 in PBS containing 1 % FBS and 0.1 % of saponin. b) a rabbit polyclonal antibody anti-nuclear TAR DNA-binding protein 43 (TDP43) at dilution of 1 / 100 in PBS containing 1 % FBS and 0.1 % of saponin.

[0143] These antibodies were revealed with a Clear Fluor (CF®) 488 goat anti-mouse IgG at the dilution 1 / 400 and a CF® 568 goat anti-rabbit IgG at the dilution 1 / 400, in PBS containing 1 % FBS, 0.1 % saponin, for 1 hour at room temperature. Nuclei were counterstained with the fluorescent dye Hoechst (Sigma Aldrich1 / 1000), a marker of total cell survival.Automatic computer analysis

[0144] For each well, 30 pictures (representative of all well area) per well were automatically taken using ImageXpress® (Molecular Devices) with 20x magnification, using the same acquisition parameters. From images, analyses were directly and automatically performed by MetaXpress® (Molecular Devices). The following endpoints were automatically assessed:- Analysis of neuron survival (MAP-2 staining, number of neurons),- Analysis of neurite network (MAP-2 staining, total neurite length in pm),- Analysis of cytoplasmic TDP43 in MAP-2 positive neurons (overlapping between MAP-2 and cytoplasmic TDP43 in pm2).

[0145] According to statistical analysis with an automatized script, 1 to 2 wells per condition can be excluded from analysis due to technical issue, without impacting the quality of results.STATISTICS

[0146] All values are expressed as mean ± SEM (standard error of the mean). Statistical analysis was performed with GraphPad Prism (version 9) using one-way ANOVA followed by Fisher’s LSD test. p< 0.05 was considered significant.Results:

[0147] Table 2 below details the results of the assay.

[0148] Table legend: Significance calculated as a significant change from glutamate insult alone, calculated using One-way ANOVA followed by Fisher's LSD test.* = P<0.05 versus glutamate** = p<o.O1 versus glutamate*** = p<0.001 versus glutamate**** > p<0.0001 versus glutamateConclusion:

[0149] This example demonstrates the effects of the test compounds in an in vitro model of ALS based on primary spinal cord MNs from SOD1 G93A transgenic animals after a glutamatergic stress. BDNF as a validated positive experimental control.

[0150] The compounds displayed neuroprotective properties with different profiles and as such evidences the use of these compounds as medicaments.EXAMPLE 3: EFFECT OF TEST COMPOUND ON ALPHA-SYNUCLEIN ENTITIES IN A PRIMARY CULTURE OF MESENCEPHALIC NEURONS INJURED WITH ALPHA-SYNUCLEIN PRE-FORMED FIBRILS (PFF)

[0151] Parkinson’s disease (PD) is the second most common neurodegenerative disorder in the United States. The predominant motor symptoms of PD, including slow movement, resting tremor, rigidity and gait disturbance, are caused by the loss of dopaminergic neurons in the substantia nigra (SN). Although the etiology of PD remains so far unknown, both genetic and environmental factors appear to play a role (Vila & Przedborski, 2004).

[0152] PD is characterized by the accumulation of intraneuronal misfolded fibrillar alpha- synuclein (aSyn) forming Lewy-bodies structures, and the degeneration of dopaminergic neurons in the SN that projects to the striatum (ST). During the progression of PD, the nigrostriatal system is severely affected. The nigrostriatal system anatomically consists of two primary structures: the putamen, or dorsal ST in rodents, and the SN, which are connected in the brain by dopamine neurons. Degeneration of the nigrostriatal dopaminergic pathways destabilizes the motor control networks.

[0153] The present example uses an in vitro model of PD injured with aSyn PFFs reproduces essential neuropathological features of PD, including loss of dopaminergic neurons, aSyn aggregation, mitochondrial and lysosomal stress (see details in Henriques et al., 2022).

[0154] The study was undertaken on one test compound at 8 concentrations. Compound 4 was tested in this in vitro model of PD induced by aSyn PFFs. Investigation of dopaminergic neuron survival (TH+), neurite network degeneration, and aSyn aggregation was assessed by ICC.MethodsPrimary culture of mesencephalic neurons

[0155] Rat dopaminergic neurons were cultured as described by Henriques et al., 2022 and Callizot et al., 2019. Briefly, a pregnant female rat (Wistar) of 15 days of gestation was killed using deep anesthesia with CO2 chamber and cervical dislocation. The midbrains obtained from 15-day-old rat embryos (Janvier, France) were dissected under a microscope. The embryonic midbrains were removed and placed in ice-cold medium of Leibovitz (L15) containing 2 % penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1 % bovine serum albumin (BSA). The ventral portion of the mesencephalic flexure, a region of the developing brain rich in dopaminergic neurons, was used for the cell preparations.

[0156] The midbrains were dissociated by trypsinization for 20 minutes at 37°C (solution at a final concentration of 0.05 % trypsin and 0.02 % EDTA). The reaction was stopped by the addition of Dulbecco’s modified Eagle medium (DMEM) containing DNase I grade II (0.5 mg / mL) and 10 % fetal calf serum (FCS). Cells were then mechanically dissociated by 3 passages through a 10 mL-pipette. Cells were then centrifuged at 180 x g for 10 minutes at +4°C on a layer of BSA (3.5 %) in L15 medium. The supernatant was discarded, and the cell pellets were re-suspended in a defined culture medium consisting of Neurobasal supplemented with B27 (2 %), L-glutamine (2 mM), 2 % PS solution, 10 ng / mL of Brain-Derived Neurotrophic Factor (BDNF) and 1 ng / mL of Glial-Derived Neurotrophic Factor (GDNF). Viable cells were counted using the LUNA-FL (logosbio) cell counter.

[0157] The cells were seeded at a density of 40,000 cells / well in 96-well plates (pre-coated with poly-L-lysine) and maintained in a humidified incubator at 37°C in 5% CO2 / 95 % air atmosphere. Half of the medium was changed every 2 days with fresh medium. The wells of the first and last rows and columns were not used (to avoid any edge effect) and were filled with sterile water.Test compounds and aSyn injury

[0158] Vehicle: Culture medium [Dimethyl sulfoxide (DMSO) 0.1 %]

[0159] aSyn preparation: Human aSyn peptide was reconstituted in medium at 4 pM (mother solution) and was slowly shaken at +37°C for 3 days in the dark to generate the aSyn PFF solution. The control medium was prepared in the same conditions.

[0160] Pre-incubation: On day 6 of culture, the test compound was solubilized in DMSO, then diluted in the culture medium and pre-incubated with primary neurons for 1 h, before injury.

[0161] Treatment: On day 6 of culture, 1 hour after the test compound / reference compounds were applied, the cells were injured with the aSyn PFF preparation (250 nM final concentration). The injury was renewed on day 8 for 48 hours, alongside the test compound / reference compounds, and the culture stopped at 96 hours post-injury.Organization of culture plate.

[0162] Test compound 4 at eight concentrations was tested on one culture in a 96-well plate (6 wells per condition). The following conditions were evaluated:End point evaluation. Immunostaining: TH and aSyn

[0163] 96 hours after injury, the cell culture supernatant was collected and discarded and the cells were fixed by a solution of 4% paraformaldehyde in PBS, pH =7.3 for 20 min at room temperature. The cells were washed twice in PBS, and then permeabilized. Non-specific sites were blocked with a solution of PBS containing 0.1 % saponin and 1 % FCS for 15 min at room temperature.

[0164] The cultures were incubated with:• Monoclonal anti-Tyrosine Hydroxylase (TH) antibody produced in mouse at a dilution of 1 / 10,000 in PBS containing 1 % FCS, 0.1 % saponin, for 2h at room temperature.• Polyclonal anti-alpha synuclein (aSyn) antibody produced in rabbit at a dilution of 1 / 200 in PBS containing 1 % FCS, 0.1 % saponin, for 2h at room temperature.

[0165] These antibodies were revealed with Alexa Fluor 488 goat anti-guinea pig IgG at the dilution 1 / 800 and with Alexa Fluor 458 goat anti-rabbit IgG secondary antibodies at the dilution 1 / 400 in PBS containing 1 % FCS, 0.1 % saponin, for 1h at room temperature.

[0166] Nuclei were counterstained with the fluorescent dye Hoechst (sigma 1 / 1000), a marker of total cell survival.Statistics

[0167] All values are expressed as mean ± SEM (standard error of the mean). Statistical analysis was performed by one-way ANOVA followed by Fisher’s LSD test. p< 0.05 was considered significant.Results

[0168] Application of aSyn (250 nM) significantly reduced dopaminergic neuronal survival (Fig. 1A), induced degeneration of the neurite network (Fig. 1 B), increased the aSyn aggregation in TH(+) neurons (Fig. 1 C), and the number of nuclei were maintained (Fig, 1 D).

[0169] Compound 4 was applied 1 hour before the injury on days 6 and 8 and used at the concentrations shown above. As shown in Figure 1 , Compound 4 markedly reduced TH(+) neuronal loss, preserved neurite network integrity and decreased aSyn aggregation. Figures1 A, 1 B and 1C demonstrate a clear concentration dependent response, with concentrations 2 and 10 nM producing the strongest effects.Conclusion

[0170] The aim of this study was to evaluate the effects of Compound 4 in an in vitro model of PD induced by aSyn PFFs. Investigation of dopaminergic neuron survival (TH+), neurite network integrity, and aSyn aggregation was assessed by ICC.

[0171] In line with the results obtained, it can be concluded that Compound 4 was able to protect the dopaminergic neurons (survival and neurites) and to reduce aSyn aggregation in these neurons in a dose dependent manner.EXAMPLE 4: EFFECTS OF TEST COMPOUNDS ON SYNAPSES IN A RAT PRIMARY CULTURE OF HIPPOCAMPAL NEURONS INJURED WITH GLUTAMATE

[0172] Alzheimer’s disease (AD) affects mainly people over the age of 65, suffering from different clinical symptoms such as progressive decline in memory, thinking, language, and learning capacity. The glutamatergic system, and in particular NMDA receptors (glutamatergic receptor) have a major role in the processes of learning and memory. Synaptic plasticity can be regulated by NMDA receptor signaling.

[0173] Excessive activation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in the pathophysiology of AD (Liu et al., 2013). Overactivation of NMDARs lead to excitotoxicity due to an increase of intracellular calcium, mitochondrial dysfunction, oxidative stress leading to neuronal cell death. Glutamate excitotoxicity and oxidative stress can be reproduced in in vitro disease by applying high concentration of glutamate on primary culture of neurons.

[0174] The aim of this study is to evaluate the effect of test compounds (tested at several concentrations) on the survival of hippocampal neurons and on the integrity of the neurite network and synapses in an in vitro model if AD induced by glutamate.Methods:Primary culture of hippocampal neurons

[0175] Rat hippocampal neurons were cultured as described by Callizot et al., 2013 with modifications. Briefly, pregnant female rat (Wistar, Janvier) of 17 days of gestation were killed using a deep anesthesia with CO2chamber and a cervical dislocation. Embryos were collected and immediately placed in ice-cold L15 Leibovitz medium with a 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA). Cortex was specifically dissected and then treated for 20 min at 37°C with a trypsin- EDTA solution at a final concentration of 0.05% trypsin and 0.02% EDTA.

[0176] The dissociation was stopped by addition of Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g / L of glucose, containing DNAse I grade II (final concentration 0.5 mg / mL) and 10% fetal calf serum (FCS). Cells were mechanically dissociated by three forced passages through the tip of a 10-mL pipette. Cells were then centrifuged at 515 x g for 10 min at 4°C. The supernatant was discarded, and the pellet was resuspended in a defined culture medium consisting of Neurobasal medium with a 2% solution of B27 supplement, 2 mM of L-glutamine, 2% of PS solution, and 10 ng / mL of brain-derived neurotrophic factor (BDNF).

[0177] Viable cells were counted using the LUNA-FL (Logos Biosystems) cell counter. The cells were seeded at a density of 20,000 per well in a 96-well plate precoated with poly-L-lysine and were cultured at 37°C in an air (95%)-CO2(5%) incubator. The medium was changed every 2 days. For 96 well-plates, only 60 wells were used. To avoid any edge effect, the first and last columns as well as first and last lines of the plate were not used in the study. Empty wells were filled with water.Test compoundsVehicle: culture medium (0.1 % DMSO).Pre-incubation: On day 17 of culture, compounds and BDNF (50 ng / mL) were dissolved in DMSO and in water, respectively, at 1000x concentration, and later at 1x concentration in the culture medium (0.1 % DMSO, final concentration). Compounds were pre-incubated with primary hippocampal neurons for 1 hour before the glutamate injury.Injury: On day 17, and 1 hour after the application of the compounds, hippocampal neurons were injured with glutamate (20 pM for 20 min), in presence of the test compound. After 20 min, glutamate was washed out and fresh culture medium with the test compound was added for an additional 48-hour period.END POINT EVALUATIONImmunostaininq: PSD95 and MAP-2

[0178] After 48 hours of injury, hippocampal neurons were fixed by a cold solution of ethanol (95 %) and acetic acid (5 %) for 5 min at -20 °C. The cells were washed twice in PBS and then permeabilized. Non-specific sites were blocked with a solution of PBS containing 0.1 % of saponin and 1 % FBS for 15 min at room temperature.

[0179] The cultures were incubated with a:• mouse monoclonal antibody anti-postsynaptic density protein 95 (PSD95, a post- synaptic marker) at dilution of 1 / 200 in PBS containing 1% FBS and 0.1 % of saponin.• chicken polyclonal antibody anti-MAP-2 (microtubule-associated protein 2), at a dilution of 1 / 400 in PBS containing 1 % FBS and 0.1 % of saponin. This antibody binds specifically neurons and neurites, allowing study of neuronal cell survival and neurite network.• Nuclei were counterstained with the fluorescent dye Hoechst H 33258 (Sigma Aldrich, 1 / 1000), a marker of total cell survival.

[0180] These antibodies were revealed with an Alexa Fluor 647 anti-chicken, an Alexa Fluor 568 anti-rabbit at a dilution of 1 / 400, and with an Alexa Fluor 488 anti-mouse IgG at the dilution 1 / 400 in PBS containing 1 % FBS, 0.1 % saponin, for 1 hour at room temperature.Automated computer analysis

[0181] For each condition, 30 pictures (representing the whole well area) were automatically taken using Operetta CLS (PerkinElmer / Revvity) with 40x magnification, using the same acquisition parameters. From images, analyses were directly and automatically performed by Harmony (PerkinElmer / Revvity).

[0182] The following endpoints were automatically assessed:- Number of PSD95 (overlapping between PSD95 / MAP-2 in pm2)- Total neuron survival (number of MAP-2 positive neurons);- Total neurite network (length of MAP-2 positive neurite in pm);- Total number of cells (nuclei counting)Statistical analysis

[0183] All values were expressed as mean + / - SEM (standard error of the mean). Results were analyzed with GraphPad Prism, version 9. Statistical analysis will be performed by one-way ANOVA, followed by LSD test. p<0.05 was considered significant.Results:

[0184] Table 3 below details the results of the assay.

[0185] Table legend: Significance calculated as a significant change from glutamate insult alone, calculated using One-way ANOVA followed by Fisher's LSD test.* = P<0.05 versus glutamate** = p<o.O1 versus glutamate*** = p<0.001 versus glutamateConclusion

[0186] The data presented in this example shows that all compounds tested were neuroprotective and able to increase neuronal plasticity.EXAMPLE 5: EFFECTS OF TEST COMPOUNDS ON NEURITE NETWORK IN A PRIMARY CULTURE OF CORTICAL NEURONS (PLASTICITY ASSAY)

[0187] Neuronal plasticity corresponds to the ability of the nervous system to reorganize itself by making new connections through neurogenesis, neuritogenesis and synaptogenesis. Lack of neuronal plasticity is found in neurodegenerative diseases, neurological disorders and during biological aging, or senescence. Low neuronal plasticity is associated with cognitive decline, depression and psychiatric diseases.

[0188] The aim of this study was to assess the effect of compounds at four different concentrations on the neurite network, roots and branching after 3 days of treatment.

[0189] Compounds were applied on primary cortical neurons, on day 1 of culture and left for 3 days. BDNF was used as a positive control.Methods:Primary culture of cortical neurons

[0190] Rat cortical neurons were cultured as described by Callizot et al., 2013 with modifications. Briefly, pregnant female rat (Wistar, Janvier) of 15 days of gestation were killed using a deep anesthesia with CO2chamber and a cervical dislocation. Fetuses were collected and immediately placed in ice-cold L15 Leibovitz medium with a 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA). Cortex wasspecifically dissected and then treated for 20 min at 37°C with a trypsin- EDTA solution at a final concentration of 0.05% trypsin and 0.02% EDTA.

[0191] The dissociation was stopped by addition of Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g / L of glucose, containing DNAse I grade II (final concentration 0.5 mg / mL) and 10% fetal calf serum (FCS). Cells were mechanically dissociated by three forced passages through the tip of a 10-mL pipette. Cells were then centrifuged at 515 x g for 10 min at 4°C. The supernatant was discarded, and the pellet was resuspended in a defined culture medium consisting of Neurobasal medium with a 2% solution of B27 supplement, 2 mM of L-glutamine, 2% of PS solution, and 10 ng / mL of brain-derived neurotrophic factor (BDNF).

[0192] Viable cells were counted using the LUNA-FL (Logos Biosystems) cell counter. The cells were seeded at a density of 25,000 per well in 96-well plate precoated with poly-L-lysine and were cultured at 37°C in an air (95%)-CO2 (5%) incubator. The medium was changed every 2 days. For 96 well-plates, only 60 wells were used. To avoid any edge effect, the first and last columns as well as first and last lines of the plate were not used in the study. Empty wells were filled with water.Test compoundsVehicle: culture medium (0.1 % DMSO).Treatment: On day 1 (day of seeding), stock solution (pure DMSO) of the compounds were diluted in the vehicle and applied on the culture and left for 3 days.END POINT EVALUATIONImmunostaininq: MAP-2

[0193] 72 hours after treatment, the cell culture supernatant was removed, and the cells were washed with phosphate-buffered saline (PBS). The cortical neurons were fixed by a cold solution of ethanol (95 %) and acetic acid (5 %) for 5 min at -20° C. They were washed twice again in PBS and then permeabilized. Non-specific sites were blocked with a solution of PBS containing 0.1 % of saponin and 1 % of FCS, for 15 min at RT. Cells were incubated for 2 hours with a mouse monoclonal antibody anti microtubule-associated-protein 2 (MAP-2), at a dilutionof 1 / 400 in PBS, with 1% fetal calf serum and 0.1% of saponin. This antibody binds specifically neurons and neurites, allowing study of neuronal cell survival and neurite network.

[0194] This antibody was revealed with a secondary antibody goat anti-mouse IgG coupled with an Alexa Fluor 488 at the dilution 1 / 400 in PBS containing 1 % FCS, 0.1 % saponin, for 1 hour at room temperature. Cell nucleus was counterstained with the fluorescent dye Hoechst (1 / 1000, Sigma-Aldrich).Automated image analysis

[0195] For each condition, pictures (representing the whole well area) were automatically taken using Operetta CLS (PerkinElmer / Revvity) with 20x magnification, using the same acquisition parameters. From images, analyses were directly and automatically performed by Hamony (PerkinElmer / Revvity).

[0196] The following endpoints were automatically assessed:- Total neurite network (length of MAP-2 positive neurite in pm)- Total of branching (extremities)- Total roots- Total number of MAP-positive cellsStatistical analysis

[0197] All values were expressed as mean + / - SEM (standard error of the mean). Results were analyzed with GraphPad Prism, version 9. Statistical analysis will be performed by one-way ANOVA, followed by LSD test. p<0.05 was considered significant.Results:

[0198] Table 4 below details the results of the assay.

[0199] Table legend: Significance calculated as a significant change from glutamate insult alone, calculated using One-way ANOVA followed by Fisher's LSD test.* = P<0.05 versus glutamate** = p<o.O1 versus glutamate*** = p<0.001 versus glutamateConclusion

[0200] The data presented in this example shows that all compounds tested were able to increase neuronal plasticity.EXAMPLE 6: EFFECT OF COMPOUND 4 AND BDNF ON TRKB PHOSPHORYLATION. IN PRIMARY CORTICAL NEURONS IN SUSPENSION

[0201] Neuronal plasticity corresponds to the ability of the nervous system to reorganize itself by making new connections through neurogenesis, neuritogenesis and synaptogenesis. Lack of neuronal plasticity is found in neurodegenerative diseases, neurological disorders and during biological aging, or senescence. Low neuronal plasticity is associated with cognitive decline, depression and psychiatric diseases. Trophic signaling, including BDNF / TrkB pathway, plays an important role in neuronal plasticity.

[0202] The aim of this study was to assess the effect of Compound 4 and BDNF on the phosphorylation of TrkB in an immature culture of primary cortical neurons in suspension. Total level of TrkB and p-TrkB was assessed by automated protein analysis.MethodsPrimary culture of cortical neurons

[0203] Rat cortical neurons were cultured as described by Callizot et al., 2021. Briefly, pregnant female rat (Wistar) of 15 days of gestation will be sacrificed using deep anesthesia with CO2 chamber and cervical dislocation.

[0204] Embryos were collected and immediately placed in ice-cold L15 Leibovitz medium with a 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1 % bovine serum albumin (BSA).

[0205] Cortices were treated for 20 minutes at 37°C with a trypsin-EDTA solution at a final concentration of 0.05 % trypsin and 0.02 % EDTA. The dissociation was stopped by the addition of Dulbecco’s modified Eagle medium (DMEM) with 4.5 g / L of glucose, containing DNAse I grade II (final concentration 0.5 mg / mL) and 10% fetal bovine serum (FBS).

[0206] Cells were mechanically dissociated by three forced passages through the tip of a 10-mL pipette. Cells were then centrifuged at 515 x g for 10 minutes at 4°C, in three distinct tubes, to allow direct re-suspension in the test vehicle. The supernatant was discarded, and the pellet resuspended in a defined medium (see below) with 10 ng / mL of brain-derived neurotrophic factor (BDNF).

[0207] Viable cells were counted using the LUNA-FL (logosbio) cell counter. An equivalent of 26,000,000 cells were collected from embryonic rat cortices and used at a density of 20,000,000 per mL at 37°C in an air (95 %)-CO2(5 %) incubator. Cells were placed in 96-well plates (20 pL / well; 400.000 cells per well) kept under agitation during treatment.Treatment

[0208] Standard Neurobasal medium + BDNF 10 ng / mL, then washing steps.Cell handling after treatment

[0209] The culture medium with cells was collected and spun down at 400 x g for 7 minutes and washed with HBSS (with compound and BDNF (10 ng / mL). Repeated twice, supernatant removed and directly proceeded to cell lysis.Organization of culture plate

[0210] Test compounds were tested on one culture in a suspension (4 wells per condition, in 96-well plates kept under agitation). The following conditions were evaluated:Sample preparation / total protein extraction and quantification

[0211] Cell pellets were lysed with a defined buffer lysis consisting of CelLyticMT reagent with 1 % of Protease and phosphatase inhibitor cocktail (50 pL per well).

[0212] For each condition, the quantity of protein was determined using the micro kit BCA (Pierce). Lysates were diluted at 1 / 20 in PBS and mixed, in equal volume, with a micro-BCA Working reagent. These solutions were incubated at 60°C for 1 hour and the quantity of protein was measured at 562 nm with a spectrophotometer Glomax Discover System (Promega) and compared with the standard of Bovin Serum Albumin curve (BSA, Pierce).Automated quantification of proteins (JESS analysis)

[0213] All reagents, (except primary antibodies), secondary antibodies, and total protein normalization module were provided by ProteinSimple®. They were prepared and used according to manufacturer’s recommendations for use of JESS™ apparatus (ProteinSimple, USA).

[0214] The run was performed according to manufacturer’s recommendations. Capillaries, samples, antibodies, and matrices were then loaded inside the instrument. The simple Western will be run with capillaries filled with separation matrix, stacking matrix and protein samples. Next, capillaries were incubated for 30 minutes with primary antibodies. Depending on the species and antibodies compatibility, two antibodies per capillary could be used. Depending on the species and antibodies compatibility, two antibodies per capillary could be used. The quantity of proteins loaded was adapted for each protein of interest and range between 0.2 and 1 .5 mg / mL. Levels of target proteins were investigated using the following primary antibodies:- Anti-TrkB (Abeam) / / a receptor of BDNF• - Anti-phospho-TrkB (Tyr816) / / the phosphorylated form of TrkB

[0215] Capillaries were washed and incubated with HRP conjugated secondary antibodies for 1 h. After removal of unbound secondary antibody, the capillaries were incubated with the luminol- S / peroxide substrate and chemiluminescent signal was collected using the Charge-Coupled Device (CCD) camera of JESS™ with six different exposure times (30, 60, 120, 240, 480, and 960 s).

[0216] After the measurements, a RePlex was performed, washing all antibodies from capillaries. Total protein reagent was loaded into capillaries labelling every amine group. Signals were collected as previously described.

[0217] Each protein was evaluated independently. Data analysis was performed using the Compass Software (ProteinSimple) on JESS™. 4 samples (biological replicates) per condition were analysed. The design did not include technical replicates. Remaining material was stored at -80°C for future investigations.

[0218] All values are expressed as mean + / - SEM (standard error of the mean). Statistical analysis was performed by one way ANOVA, followed by a Fisher’s LSD test. p<0.05 will be considered significant.Results

[0219] Figure 2 details the data obtained from this experiment. As can be seen BDNF statistically significantly increased the ratio of pTrKB to TrkB in a culture of primary cortical neurons in suspension.

[0220] Similarly, Compound 4 at 10 pM was also found to statistically significantly increase the ratio of pTrKB to TrkB.Conclusion

[0221] These data confirm that Compound 4 was able to increase pTrkB to TrkB ratio in a culture of primary cortical neurons in suspension, suggesting that this compound activates TrkB.EXAMPLE 7: EFFECT OF TEST COMPOUNDS IN A NEURONAL PLASTICITY ASSAY OF NEURITE NETWORS IN A PRIMARY CULTURE OF CORTICAL NEURONS

[0222] Neuronal plasticity corresponds to the ability of the nervous system to reorganize itself by making new connections through neurogenesis, neuritogenesis and synaptogenesis. A lack of neuronal plasticity is found in neurodegenerative diseases, neurological disorders and during biological aging, or senescence. Low neuronal plasticity is associated with cognitive decline, depression and psychiatric diseases.

[0223] The aim of this study was to assess the effect of several compounds on the neurite network, roots and branching after 3 days of treatment. The test compound was applied on primary cortical neurons, on day 1 of culture, and incubated for 3 days. BDNF was used (at 1 concentration) as a positive control. In addition, several psychedelic compounds and ketamine, a NMDA receptor antagonist were tested and compared to the test compounds.MethodsPrimary culture of cortical neurons

[0224] Rat cortical neurons were cultured as follows. Pregnant female rats of 15 days gestation (Rats Wistar; Janvier Labs France) were euthanised under deep anesthesia using CO2and cervical dislocation. Embryos were then removed from the uterus and immediately placed in ice- cold L15 Leibovitz medium with 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA).

[0225] Cortical samples were treated for 20 minutes at 37°C with trypsin-EDTA solution at a final concentration of 0.05% trypsin and 0.02% EDTA. The dissociation was stopped by addition of Dulbecco’s modified Eagle medium (DMEM) with 4.5 g / L of glucose, containing DNAse I grade II (final concentration 0.5 mg / mL) and 10% fetal bovine serum (FBS).

[0226] Cells were mechanically dissociated by three forced passages through the tip of a 10-mL pipette. Cells were then centrifuged at 515 x g for 10 minutes at 4°C. The supernatant was discarded, and the pellet resuspended in a defined culture medium consisting of Neurobasal medium with a 2% solution of B27 supplement, 2 mM of L-glutamine, 2% of PS solution, and 10 ng / mL of brain-derived neurotrophic factor (BDNF).

[0227] Viable cells were counted using the LUNA-FL (Logos Biosystems) cell counter. Cells were seeded at a density of 25,000 per well in 96-well plates precoated with poly-L-lysine and cultured at 37°C in an air (95%)-CO2 (5%) incubator. The medium was renewed every other day. To avoid any edge effect, the first and last columns as well as first and last rows of the plate were not used in the study. Empty wells were filled with sterile water.Test compounds

[0228] Vehicle: Culture medium (0.1% DMSO, 0.1 % acetonitrile, 0.1% MeOH).

[0229] Treatment: On day 1 (day of seeding), stock solutions (pure DMSO, acetonitrile or MeOH) of the compounds will be diluted in the vehicle, applied on the culture and incubated for 3 days.

[0230] Positive control: BDNF (Brain-derived neurotrophic factor).

[0231] Test compounds: CF3CN (4-(6-oxo-2-(trifluoromethyl)-3,6-dihydrochromeno[7,8- d]imidazol-8-yl)benzonitrile); Compound 2; Compound 4; and Compound 6.

[0232] Psychedelic compounds: Psilocin; LSD (lysergic acid diethylamide); 5-MeO-DMT (5- methoxy-N,N-dimethyltryptamine).

[0233] NMDA receptor antagonist: ketamine.Organization of culture plate

[0234] Test compounds will be tested on one culture in 96-well plate (n = 6 culture wells per condition). The following conditions will be assessed:End point evaluationImmunostaininq: MAP2

[0235] 72 hours after injury, the cell culture supernatant was removed, and cells were fixed using a cold solution of ethanol (95%) and acetic acid (5%) for 5 minutes at -20°C. The cells were washed twice in Phosphate-buffered saline (PBS). Cell membranes were permeabilized and non-specific binding sites blocked with a solution of PBS containing 0.1% saponin and 1% FBS for 15 minutes at room temperature.

[0236] The following antibody was applied for 2 hours at room temperature -MAP2 neurons: Mouse monoclonal anti-microtubule-associated-protein 2 (MAP2) antibody at a dilution of 1 / 400, in PBS containing 1 % FBS, 0.1 % saponin.

[0237] This primary antibody was revealed with an Alexa Fluor 488 anti-mouse IgG at the dilution 1 / 400, in PBS containing 1 % FBS, 0.1 % saponin, for 1 hour at room temperature. Nuclei will be counterstained with the fluorescent dye Hoechst (Sigma-Aldrich, 1 / 1000): marker of cell number.Automatic computer analysis

[0238] For each condition, 25 pictures (representing the whole well area) were automatically taken using Operetta (High-Content Analysis System Design, Revvity) at 20x magnificationusing the same acquisition parameters. From images, analyses will be automatically performed by Harmony (Revvity).

[0239] The following read-outs were measured:- Total neurite network (length of MAP2(+) neurites in pm).- Total of branching points of neurites (extremities).- Total roots of neurites.- Total number of neurons (MAP2(+) cells

[0240] A representative image of automated morphological analysis by Harmony analysis software can be seen in Figure 3.Statistical analysis

[0241] All values were expressed as mean ± SEM (standard error of the mean). Statistical analysis was performed by one-way ANOVA, followed by a Fisher’s LSD test, p < 0.05 was considered significant.

[0242] EC5O values were calculated using GraphPad PrismCriteria of validation

[0243] The experiment / the culture plates were only validated if the application of the BDNF (50 ng / mL, all plates) resulted in a significant effect on the neurite network, and on the number of branching points and roots, when compared to the control condition (One-way ANOVA followed by Fisher's LSD).Results

[0244] Plates 1 and 2 made head-to-head comparisons at concentrations estimated to drive maximal effect on neuritogenesis (informed by historical data).

[0245] Plates 3-7 contained concentration-response curves for ketamine, psilocin, LSD, CF3CN and Compound 4.

[0246] Figures 4 to 12 detail the analysis of the individual plates as detailed above for all four read outs.

[0247] All compounds increase neuritogenesis in primary rat cortical cultures as measured by neurite network length (primary endpoint)

[0248] Figure 11 demonstrates that the test compounds, Compounds 2, 4 and 6 all promoted similar maximum neuritogenesis effects as ketamine and psychedelics in the head-to-head experiments.

[0249] Figure 12 and Table 5 below detail the EC5o values obtained from the concentration curve data for Psilocin, LSD, Ketamine, CF3CN and Compound 4. These data demonstrate that there was a strong concentration-dependent increase in neuritogenesis seen for all compounds. In addition, both CF3CN and Compound 4 were more potent than ketamine and psychedelics in promoting neuritogenesis.Table 5. EC5o values for the test compoundsDiscussion

[0250] The aim of this study was to evaluate the effects of the test compounds on neuronal plasticity. Compounds such as ketamine and psychedelics are known to enable the nervous system to reorganize itself by making new connections through neurogenesis, neuritogenesis and synaptogenesis.

[0251] The data obtained by this example surprisingly demonstrated that the compounds of the invention were able to promote neuronal plasticity, indeed their effect was found to be morepotent than that of known neuritogenesis promoters such as ketamine and psychedelics. Thus, suggesting that these compounds may be efficacious in the treatment of diseases and disorders where neuronal plasticity is lacking or decreased such as neurodegenerative diseases, neurological disorders, biological aging, senescence, cognitive decline, depression and psychiatric diseases.EXAMPLE 8: EFFECTS OF TEST COMPOUNDS IN CHRONIC SOCIAL DEFEAT STRESS INDUCED DEPRESSION MOUSE MODEL

[0252] The object of this study was to determine the efficacy of the test compounds in a chronic social defeat stress induced depression mouse model. Fluoxetine was used as a positive control.MethodsAnimals

[0253] C57BL / 6j mice (male, 7 weeks old) and GDI mice (male, 9-10 weeks old) were acclimatised for at least 1 week prior to the experiment.

[0254] Animals were kept under standard conditions with room temperature 21-23°C, 40-70% RT and a 12 / 12h light / dark cycle. Chow and water were freely available.Test articles

[0255] Fluoxetine and test articles were prepared for each dosing as follows.

[0256] Vehicle: 0.5% methylcellulose (400cP). 5.0g of methylcellulose was weighed into 1000ml of distilled water and stirred for 2h to obtain a clear solution which was stored at 4°C.

[0257] Vehicle (5% DMSO + 95% water (0.5% methylcellulose (400cP)): 50ml DMSO was added to 0.5% methylcellulose to 1 .00L and mixed well by vigorous vortex and stirred to obtain a clear solution and stored at 4°C.

[0258] Fluoxetine: 5.13mg of fluoxetine was weighed and added to 4.5ml of saline and mixed well by vigorous vortex and sonication.

[0259] Test compounds: 4.94ml of DMSO was added to 200.1 mg of test article and mixed well by vigorous vortex and sonication.Social avoidance test

[0260] On day 11 a social avoidance test was performed to identify subgroups of mice that were susceptible to and unsusceptible to social defeat stress.

[0261] This was accomplished by placing mice in an interaction test box (42x42cm) with an empty wire mesh cage (10x4.5cm) located at one end.

[0262] The movement of the mice was tracked for 2.5min followed by 2.5min in the presence of an unfamiliar aggressor GDI mouse in the wire mesh cage.

[0263] The duration of the subject mouse’s presence in the interaction zone (an 8cm wide area surrounding the wire mesh cage) was recorded by software (Model SA224, SANS).

[0264] The interaction ratio was calculated as time spent in an interaction zone with an aggressor / time spent in an interaction zone without an aggressor.

[0265] An interaction ratio of 1 was set as the cut off: mice with scores <1 were defined as susceptible to social defeat stress and were included in the study, and those with scores >1 were defined as unsusceptible.Social interaction test (SIT)

[0266] An apparatus of total size 60x45x45cm was divided into three communicating chambers with openings between the compartments allowing the mice to move freely between. Wire cages were placed in each of the lateral compartments.

[0267] The study consisted of three phases: habituation phase; sociability phase; and social novelty preference test.

[0268] Sociability Index (SI) and social novelty preference (SNP) were calculated.

[0269] The test mouse was first placed in the middle chamber and allowed to explore with the doorways to the two side chambers wide open.

[0270] After the habituation period the test mouse was enclosed in the centre compartment and an unfamiliar mouse (stranger 11; a male C57BL / 6j adult mouse) was enclosed in a wire cage and placed in a side chamber. All stranger mice had previously been habituated to the wire cage.

[0271] The location for stranger 1 alternated between the left and right side of the social test box. An empty wire cage was placed in the opposite side to serve as a novel object control.

[0272] Following placement of stranger 1 , the doors were re-opened and the subject was allowed to explore the entire social test box and the time spent in each chamber and the number of entries into each chamber were recorded via the automated test system.

[0273] Sociability Index (SI)=(Total duration of contacts in stranger 1 side - Total duration of contacts in empty cage side) / (Total duration of contacts in stranger 1 side +Total duration of contacts in empty cage side).Preference for social novelty

[0274] At the end of the sociability test, each mouse was further tested for preference for time spent with a more novel stranger.

[0275] A new unfamiliar mouse (stranger 2) was placed in the wire cage that had been empty during the previous session. The test mouse then had a choice between the first already- investigated mouse (stranger 1) and the novel unfamiliar mouse (stranger 2).

[0276] The same measures were taken as the sociability test.

[0277] Social Novelty Index (SNI)=(Total duration of contacts in stranger 2 side - Total duration of contacts in stranger 1 side) / (Total duration of contacts in stranger 2 side +Total duration of contacts in stranger 1 side).Forced swim test

[0278] Mice were individually placed in an open cylindrical container filled with water up to a height of 20cm and forced to swim for 6min. The amount of time spent by the mice floating in the water without struggling and escape-orientated behaviour were defined as immobility time.

[0279] The duration of immobility was recorded and analysed automatically by software (Model SA209, SANS) for the last 5 min.Tissue collection

[0280] On day 34, 30 min post compound treatment animals were euthanized with CO2. Blood, plasma and brains were collected and perfused intracardially with ice-cold saline and stored at - 80°C.Statistical analysis

[0281] All statistical analysis were performed using GraphPad Prism Version 7.0. Data was analysed by One Way ANOVA followed by Dunnett’s multiple comparison or a repeated measures Two Way ANOVA followed by Dunnett’s multiple comparison.Results

[0282] Figures 13 to 15 detail the results obtained from the social interaction test for sociability and social novelty and the forced swim test.

[0283] As can be seen, all test compounds at all concentrations were able to rescue the deficits produced by the chronic social defeat stress model.Conclusion

[0284] These data provide evidence of the potential for these compounds to be developed as medicaments.

Claims

CLAIMS1 . A compound as defined by any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof.

2. A pharmaceutical composition comprising a compound of any one of the compounds numbered 1 to 8 in Table 1 , or a salt thereof, together with one or more ingredients selected from carriers, diluents, excipients, adjuvants, fillers, buffers, binders, disintegrants, preservatives, antioxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.

3. A compound of any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof or a pharmaceutical composition comprising the compound any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof, for use as a medicament.

4. A method of treatment comprising administering to a subject in need of treatment a therapeutically effective amount of a compound of any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof.

5. A method of synthesizing a compound of any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof.

6. An intermediate formed in the method of synthesis of the compound of any one of the compounds numbered 1 to 8 in Table 1 or a salt thereof.

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