Multi-target directed ligands for alzheimer's disease

Abstract

Multi-target ligands, their synthesis, and their use in treating neurodegenerative diseases are provided.

Description

Dkt.93597 / 7185 92150-A-PCT MULTI-TARGET DIRECTED LIGANDS FOR ALZHEIMER’S DISEASE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No.63 / 537,004, filed September 7, 2023, the contents of which are hereby incorporated by reference. BACKGROUND

[0002] The disclosures of all publications, patents, patent application publications and books referred to in this application are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0003] Alzheimer’s Disease (AD) is a progressive neurodegenerative disease with a life expectancy of 4-10 years following diagnosis.[1–3] As the mortality rate of other diseases reduces with the drug discovery efforts, AD prevalence increases and strikes as a leading cause of death in aging population.[4,5] Aside from the detrimental effect on patient’s daily life, the disease is also an economic burden in terms of healthcare costs.[4,6] Although it has been over a century since the disease was characterized, originating disease pathology remains to be elucidated.[4] Hence, many hypotheses have been put forward to elucidate the disease mechanism and develop disease- modifying treatments (DMT) and early diagnosis tools. The hypotheses are mainly based on pathological hallmarks, including amyloid beta-42 (Aβ42) deposition, their aggregation as plaques, tau hyperphosphorylation and their aggregates as tangles, identified over a century ago. Others include the metal-ion hypothesis, autophagy, and inflammation.[7,8] The drug discovery studies focusing single hypothesis failed to provide a DMT. So far, the only treatment available is to relieve symptoms.[4] Aducanumab, a DMT agent primarily acts on Aβ42 deposition, recently approved by the Food and Drug Administration (FDA). However, its efficacy and safety is still a concern and its annual listing price avoids widespread use.[9,10] Lecanemab provides a better DMT profile yet longitudinal effects are unclear and patients with vascular deposition of amyloid are at risk for brain hemorrhage

[0011] . AD is a complex disease where many pathological mechanisms act in concert and eventually result in neurodegeneration. Therefore, pharmacological inhibition of newer targets that display a key role that compensates various hypotheses is a promising approach for the treatment of AD. 4894-7263-3313v.1SUMMARY OF THE INVENTION

[0004] A compound represented by the formulaR is , is ‒CH3, ‒CH(CH3)2, ‒C(CH3)3,‒CH2(CH2)2CH3, ‒CH2CH2N(CH3)2, ‒CH2CH2CH2N(CH3)2, 2- tetrahydrofurylmethyl, cyclohexyl, cyclohexylmethyl, ‒CH2CH2N(CH(CH3)2)2, 2- pyridyl, 4-pyridyl, or 2-pyrimidyl; and wherein: ,4894-7263-3313v.1or a pharmaceutically acceptable salt thereof.

[0005] A pharmaceutical composition comprising a compound described herein.

[0006] A method for treating a neurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of compound described herein or an effective amount of a pharmaceutical composition comprising a compound described herein.

[0007] A method for preventing neurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of compound described herein or an effective amount of a pharmaceutical composition comprising a compound described herein.

[0008] A method for producing a compound described herein comprising the steps of: reacting with thiocyanogen to ;wherein: 3 4894-7263-3313v.1R is , wherein R’ is ‒CH3, ‒CH(CH3)2, ‒C(CH3)3, ‒CH2(CH2)2CH3, ‒CH2C2CH2N(CH3)2, 2-tetrahydrofurylmethyl, cyclohexyl, cyclohexylmethyl, ‒CH2CH2N(CH(CH3)2)2, 2-pyridyl, 4-pyridyl, or 2-pyrimidyl; and wherein: ,the method comprising the steps of: Rreacting Ar‒NH2 with 3-chloropropionyl chloride to obtain Ar‒NH‒CO‒(CH2)2‒Cl; and to .4 4894-7263-3313v.1BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIGS. 1(A)-1(C): (A) Structure-activity relationship of P2X7R antagonists. (B) Discovery pipeline of potent ChEI-based lead MTDLs. (C) Excluded hit MTDLs and aim of the lead optimization studies.

[0011] FIG. 2: Lead optimization strategy for ChEI-based MTDLs to enhance acetylcholinesterase inhibition, improved solubility and barrier penetration rate.

[0012] FIG.3: An exemplary synthesis pathway.

[0013] FIGS.4(A)-4(C): In vivo neuroprotective activity of newly generated and parent ChEI- based MTDLs on adult zebrafish AD model. (A) Graphical representation of the cerebroventricular microinjection and experimental scheme in adult zebrafish brain. (B) Illustrative images for SV2 (synaptic connections) immunohistochemistry in fish brains injected with vehicle + phosphate- buffered saline (PBS), vehicle + Aβ42, newly generated and parent MTDLs + Aβ42. Scale bars indicate 20 μm. SV2: green, DAPI: blue. (C) Quantification graph for the relative synaptic density. Ctrl: group treated with 0.1% DMSO and vehicle; Aβ4220 μM; DPZ: donepezil (10 μM); MTDLs 7, 6-7, and 5,6-7 were tested at 10 μM concentration. The statistical analysis was evaluated using one-way ANOVA; in comparison with Aβ42-treated control levels (black), in comparison with donepezil (orange), in comparison with vehicle control (blue) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0014] FIGS.5(A)-5(C): In vivo neuroprotective activity of newly generated and parent ChEI- based MTDLs on adult zebrafish AD model. (A) Graphical representation of the cerebroventricular microinjection and experimental scheme in adult zebrafish brain. (B) Illustrative images for SV2 (synaptic connections) immunohistochemistry in fish brains injected with vehicle + phosphate- buffered saline (PBS), vehicle + Aβ42, newly generated and parent MTDLs + Aβ42. Scale bars indicate 20 μm. SV2: green, DAPI: blue. (C) Quantification graph for the relative synaptic density. Ctrl: group treated with 0.1% DMSO and vehicle; Aβ4220 μM; DPZ: donepezil (10 μM); MTDLs 15, 6-15, and 5,6-15 were tested at 10 μM concentration. The statistical analysis was evaluated using one-way ANOVA; in comparison with Aβ42-treated control levels (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0015] FIGS.6(A)-6(C): In vivo neuroprotective activity of newly generated and parent ChEI- based MTDLs on adult zebrafish AD model. (A) Graphical representation of the cerebroventricular 5 4894-7263-3313v.1microinjection and experimental scheme in adult zebrafish brain. (B) Illustrative images for SV2 (synaptic connections) immunohistochemistry in fish brains injected with vehicle + phosphate- buffered saline (PBS), vehicle + Aβ42, newly generated and parent MTDLs + Aβ42. Scale bars indicate 20 μm. SV2: green, DAPI: blue. (C) Quantification graph for the relative synaptic density. Ctrl: group treated with 0.1% DMSO and vehicle; Aβ4220 μM; DPZ: donepezil (10 μM); MTDLs 16, 6-16, and 5,6-16 were tested at 10 μM concentration. The statistical analysis was evaluated using one-way ANOVA; in comparison with Aβ42-treated control levels (black), in comparison with donepezil (orange) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0016] FIGS.7(A)-7(D): In vitro cytotoxicity of newly generated ChEI-based MTDLs, 6-7, 6- 15, 6-16, 5,6-15, and 5,6-16 on 10 weeks old 3D cultures with pHAs. (A) Representative images for AceTub:red, GFAP:yellow immunocytochemical staining in 3D cultures of pHAs treated with vehicle (0.1% DMSO) and five ChEI-based MTDLs. Scale bars indicate 50 μm. DAPI: blue. Quantification graph for total number of DAPI per hydrogel (B); total GFAP volume μm3 / DAPI per hydrogel (C); total AceTub μm3 / DAPI per hydrogel (D); Ctrl: group treated with 0.1% DMSO; DPZ: donepezil (1 μM); MTDLs 6-15, 6-16, 5,6-16 were tested at 5 μM; 6-7 and 5,6-15 were tested at 1 μM concentration. The statistical analysis was evaluated using one-way ANOVA; in comparison with control levels (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0017] FIGS. 8(A)-8(C): In vitro cytoprotective activity of newly generated ChEI-based MTDLs, 6-7, 6-15, 6-16, 5,6-15, and 5,6-16 on 10 weeks of 3D cultures with Aβ42-treated pHAs. (A) Representative images for AceTub:red, GFAP:yellow immunocytochemical staining in 3D cultures of Aβ42 treated pHAs treated with vehicle (0.1% DMSO) and five new ChEI-based MTDLs. Scale bars indicate 50 μm. Aβ42:white, DAPI: blue. (B) Quantification graph for total AceTub filament length μm / DAPI per hydrogel. (C) Quantification graph for total GFAP filament length μm / DAPI per hydrogel. Ctrl without (wo) Aβ42: group treated with 0.1% DMSO; Ctrl with (w) Aβ42: group treated with 0.1% DMSO; DPZ: donepezil (1 μM); MTDLs 6-15, 6-16, 5,6-16 were tested at 5 μM; 6-7 and 5,6-15 were tested at 1 μM concentration. The statistical analysis was evaluated using one-way ANOVA; in comparison with Aβ42 control levels (black), in comparison with control levels (green) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0018] FIG.9: Examples of compounds, MTDL 1, 2 and 3.

[0019] FIG.10: Examples of synthetic pathways.

[0020] FIG.11: Pharmacokinetics study protocol. 6 4894-7263-3313v.1

[0021] FIGS.12(A) – 12(G): The effect of multi-target directed ligand (MTDL) treatment on cognitive performance, synaptic integrity, and neuropathology in APP-KI mice. (A) Heatmaps showing the time spent by mice in different arms of the T-maze. Control vehicle mice spend significant time in the retention arm, while APP-KI vehicle mice show reduced preference for the acquisition arm. MTDL treatment (MTDL 1, MTDL 2, MTDL 3) increases the time spent in the acquisition arm, indicating improved memory performance. (B) Quantification of the T-maze performance, with individual dots representing the performance of each mouse. The left plot shows the time spent in the acquisition arm (in seconds), and the right plot shows the T4 / T5 time ratio, reflecting the novel arm exploration. MTDL treatments significantly improve these metrics compared to APP-KI vehicle mice. (C) Heatmaps showing the time spent by mice around the novel and identical objects in the Novel Object Location (NOL) test. Control vehicle mice show a preference for the novel object location, while APP-KI vehicle mice exhibit impaired discrimination. MTDL treatment restores the preference for the novel object. (D) Quantification of the discrimination index (DI) from the NOL test. Each dot represents an individual mouse. MTDL treatment significantly improves the DI compared to APP-KI vehicle mice, bringing it closer to control levels. (E) Representative trajectories of mice in the T-maze, showing the path taken during the testing period. Control vehicle mice display organized exploration patterns, while APP-KI vehicle mice exhibit more erratic movements. MTDL treatment leads to a more structured exploration pattern, similar to control mice. (F) Immunofluorescence images showing synaptophysin (green) and DAPI (blue) staining in entorhinal cortex brain sections from control, APP-KI, and APP-KI MTDL-treated mice. Synaptic density appears reduced in APP-KI vehicle mice, while MTDL treatment restores synaptic puncta to levels comparable to control. (G) Quantification of synaptophysin puncta. The left plot shows the surface area of synaptophysin puncta, and the right plot shows the number of puncta per unit area. MTDL treatment significantly increases both parameters compared to APP-KI vehicle mice, indicating restoration of synaptic integrity. DETAILED DESCRIPTION

[0022] A compound represented by the formula 7 4894-7263-3313v.1, ‒CH3, ‒CH(CH3)2, ‒C(CH3)3, ‒CH2(CH2)2CH3, ‒CH2CH2N(CH3)2, ‒CH2CH2CH2N(CH3)2, 2- tetrahydrofurylmethyl, cyclohexyl, cyclohexylmethyl, ‒ CH2CH2N(CH(CH3)2)2, 2-pyridyl, 4-pyridyl, or 2-pyrimidyl; and wherein: ,8 4894-7263-3313v.1

[0023] In embodiments, the compound is represented by the formulaR1is ‒H or ‒OCH3, and R is ‒CH2(CH2)2CH3, ‒CH2(CH2)4CH3, cyclohexyl, or cyclohexylmethyl; or R1is ‒OCH3and R is 4-t-butylbenzyl, or a pharmaceutically acceptable salt thereof.

[0024] In embodiments, the compound is represented by the formula

[0025] In embodiments, the compound is represented by the formula

[0026] In embodiments, the compound is represented by the formula

[0027] In some embodiments, 2-tetrahydrofurylmethyl is of (R)-configuration. 9 4894-7263-3313v.1

[0028] In some embodiments, 2-tetrahydrofurylmethyl is of (S)-configuration.

[0029] A pharmaceutical composition comprising a compound described herein and a pharmaceutical excipient.

[0030] A method for treating a neurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of compound described herein or an effective amount of a pharmaceutical composition comprising a compound described herein.

[0031] In embodiments, treating the disease comprises improving one or more cognitive performance measures.

[0032] A method for preventing neurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of compound described herein or an effective amount of a pharmaceutical composition comprising a compound described herein.

[0033] A method of improving synaptic integrity in a subject having an impaired synaptic integrity associated with a neurodegenerative disease, the method comprising the step of administering to a subject in need thereof an effective amount of a compound described herein or an effective amount of the pharmaceutical composition described herein.

[0034] In embodiments, administering the effective amount of the compound or composition effects an increase in surface area of and / or number of synaptic puncta.

[0035] In embodiments of the methods, the neurodegenerative disease is Alzheimer’s disease.

[0036] A method for producing a compound described herein comprising the steps of: reacting with thiocyanogen to obtain;reacting with 3-chloropropionyl chloride to obtain10 4894-7263-3313v.1ainthe method comprising the steps of: R reactingthiocyanogento reacting Ar‒NH2with 3-chloropropionyl chloride to obtain Ar‒NH‒CO‒(CH2)2‒Cl; and 11 4894-7263-3313v.1reacting Ar‒NH‒CO‒(CH2)2‒Cl with .

[0038] In embodiments, thethiocyanate.

[0039] In some embodiments, a pharmaceutically acceptable salt of any of the above compounds is provided. In some embodiments, an ester of any of the above compounds is provided. In some embodiments, an enantiomer of any of the above compounds is provided.

[0040] In some embodiments, a package or composition comprising any of the above compounds is provided.

[0041] In some embodiments, a salt of the compound of the present invention is used in any of the above methods, uses, packages or compositions. In some embodiments, a pharmaceutically acceptable salt of the compound of the present invention is used in any of the above methods, uses, packages or compositions. In some embodiments, an ester of the compound of the present invention is used in any of the above methods, uses, packages or compositions. Any of the above compounds may be used in any of the disclosed methods, uses, packages or pharmaceutical compositions.

[0042] Any of the compounds used in the disclosed methods, uses, packages or pharmaceutical compositions may be replaced with any other compound disclosed in the present invention. Any of the above generic compounds may be used in any of the disclosed methods, uses, packages or compositions.

[0043] Except where otherwise specified, where the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, a scalemic mixture or isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in "Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column. 12 4894-7263-3313v.1

[0044] The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

[0045] It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as 12C, 13C, or 14C. Furthermore, any compounds containing 13C or 14C may specifically have the structure of any of the compounds disclosed herein.

[0046] It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as 1H, 2H (D), or 3H (T). Furthermore, any compounds containing 2H or 3H may specifically have the structure of any of the compounds disclosed herein.

[0047] Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.

[0048] Deuterium (2H or D) is a stable, non-radioactive isotope of hydrogen and has an atomic weight of 2.0144. Hydrogen atom in a compound naturally occurs as a mixture of the isotopes 1H (hydrogen or protium), D (2H or deuterium), and T (3H or tritium). The natural abundance of deuterium is 0.0156%. Thus, a compound with a level of deuterium at any site of hydrogen atom in the compound that has been enriched to be greater than its natural abundance of 0.0156%, is novel over its non-enriched counterpart.

[0049] In the compounds used in the method of the present invention, the substituents may be substituted or unsubstituted, unless specifically defined otherwise.

[0050] In the compounds used in the method of the present invention, alkyl, heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

[0051] It is understood that substituents and substitution patterns on the compounds used in the method of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than 13 4894-7263-3313v.1one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0052] In choosing the compounds used in the method of the present invention, one of ordinary skill in the art will recognize that the various substituents, e.g., R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.

[0053] As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Thus, C1- Cn as in “C1–Cn alkyl" is defined to include groups having 1, 2......, n-1 or n carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, isopropyl, isobutyl, sec-butyl and so on. An embodiment can be C1-C12 alkyl, C2-C12 alkyl, C3-C12 alkyl, C4-C12 alkyl and so on. An embodiment can be C1-C8 alkyl, C2-C8 alkyl, C3-C8 alkyl, C4-C8 alkyl and so on. “Alkoxy" represents an alkyl group as described above attached through an oxygen bridge.

[0054] The term "alkenyl" refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non-aromatic carbon-carbon double bonds may be present. Thus, C2-Cn alkenyl is defined to include groups having 1, 2...., n-1 or n carbons. For example, "C2-C6 alkenyl" means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and at least 1 carbon-carbon double bond, and up to, for example, 3 carbon-carbon double bonds in the case of a C6 alkenyl, respectively. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. As described above with respect to alkyl, the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated. An embodiment can be C2-C12 alkenyl or C2-C8 alkenyl.

[0055] The term "alkynyl" refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present. Thus, C2-Cn alkynyl is defined to include groups having 1, 2...., n-1 or n carbons. For example, "C2-C6 alkynyl" means an alkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl and butynyl. As described above with respect to alkyl, the straight or branched portion of the alkynyl group may contain triple bonds and may be 14 4894-7263-3313v.1substituted if a substituted alkynyl group is indicated. An embodiment can be a C2-Cn alkynyl. An embodiment can be C2-C12 alkynyl or C3-C8 alkynyl.

[0056] As used herein, "hydroxyalkyl" includes alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an -OH group. In some embodiments, C1-C12 hydroxyalkyl or C1-C6 hydroxyalkyl. C1-Cn as in “C1–Cn alkyl" is defined to include groups having 1, 2, ...., n-1 or n carbons in a linear or branched arrangement (e.g. C1–C2 hydroxyalkyl, C1–C3 hydroxyalkyl, C1–C4 hydroxyalkyl, C1–C5 hydroxyalkyl, or C1–C6 hydroxyalkyl) For example, C1–C6, as in "C1–C6 hydroxyalkyl" is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched alkyl arrangement wherein a hydrogen contained therein is replaced by a bond to an -OH group.

[0057] As used herein, "heteroalkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and at least 1 heteroatom within the chain or branch.

[0058] As used herein, "monocycle" includes any stable polyatomic carbon ring of up to 10 atoms and may be unsubstituted or substituted. Examples of such non-aromatic monocycle elements include but are not limited to: cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of such aromatic monocycle elements include but are not limited to: phenyl.

[0059] As used herein, "bicycle" includes any stable polyatomic carbon ring of up to 10 atoms that is fused to a polyatomic carbon ring of up to 10 atoms with each ring being independently unsubstituted or substituted. Examples of such non-aromatic bicycle elements include but are not limited to: decahydronaphthalene. Examples of such aromatic bicycle elements include but are not limited to: naphthalene.

[0060] As used herein, "aryl" is intended to mean any stable monocyclic, bicyclic or polycyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be unsubstituted or substituted. Examples of such aryl elements include but are not limited to: phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.

[0061] The term "heteroaryl", as used herein, represents a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Bicyclic aromatic 15 4894-7263-3313v.1heteroaryl groups include phenyl, pyridine, pyrimidine or pyridazine rings that are (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S. Heteroaryl groups within the scope of this definition include but are not limited to: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl, hexahydroazepinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetra-hydroquinoline. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.

[0062] The term "heterocycle", “heterocyclyl” or “heterocyclic” refers to a mono- or poly- cyclic ring system which can be saturated or contains one or more degrees of unsaturation and contains one or more heteroatoms. Preferred heteroatoms include N, O, and / or S, including N- oxides, sulfur oxides, and dioxides. Preferably the ring is three to ten-membered and is either saturated or has one or more degrees of unsaturation. The heterocycle may be unsubstituted or 16 4894-7263-3313v.1substituted, with multiple degrees of substitution being allowed. Such rings may be optionally fused to one or more of another "heterocyclic" ring(s), heteroaryl ring(s), aryl ring(s), or cycloalkyl ring(s). Examples of heterocycles include, but are not limited to, tetrahydrofuran, pyran, 1,4- dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1,3-oxathiolane, and the like.

[0063] The term “ester” is intended to a mean an organic compound containing the R-O-CO- R’ group.

[0064] The term “substitution”, “substituted” and “substituent” refers to a functional group as described above in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms, provided that normal valencies are maintained and that the substitution results in a stable compound. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Examples of substituent groups include the functional groups described above, and halogens (i.e., F, Cl, Br, and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4- trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulfanyl groups, such as methylsulfanyl, ethylsulfanyl and propylsulfanyl; cyano; amino groups, such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and carboxyl. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By independently substituted, it is meant that the (two or more) substituents can be the same or different.

[0065] The compounds used in the method of the present invention may be prepared by techniques known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0066] Compounds used in the method of the present invention may be prepared by techniques described in Vogel’s Textbook of Practical Organic Chemistry, A.I. Vogel, A.R. Tatchell, B.S. 17 4894-7263-3313v.1Furnis, A.J. Hannaford, P.W.G. Smith, (Prentice Hall) 5th Edition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5th Edition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0067] The various R groups attached to the aromatic rings of the compounds disclosed herein may be added to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reactions and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.

[0068] Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition.

[0069] As used herein, the term “pharmaceutically active agent” means any substance or compound suitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, substances and compounds described in the Physicians’ Desk Reference (PDR Network, LLC; 64th edition; November 15, 2009) and “Approved Drug Products with Therapeutic Equivalence Evaluations” (U.S. Department Of Health And Human Services, 30th edition, 2010), which are hereby incorporated by reference. Pharmaceutically active agents which have pendant carboxylic acid groups may be modified in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Where a pharmaceutically active agent does not possess a carboxylic acid group, the ordinarily skilled artisan will be able to design and incorporate a carboxylic acid group into the pharmaceutically active agent where esterification may subsequently be carried out so long as the modification does not interfere with the pharmaceutically active agent’s biological activity or effect.

[0070] The compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used to treat an infection or disease caused by a pathogen, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such 18 4894-7263-3313v.1as amines; alkali or organic salts of acidic residues such as phenols; alkali or organic salts of acidic residues such as carboxylic acids. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkali metal salts, sodium, potassium or lithium. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.66:1-19).

[0071] As used herein, "treating" means inhibiting, slowing, halting, or reversing the progression of a disease or infection. Treating may also mean improving one or more symptoms of a disease or infection.

[0072] The compounds used in the method of the present invention may be administered in various forms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds. This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.

[0073] The dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect. 19 4894-7263-3313v.1

[0074] A dosage unit of the compounds used in the method of the present invention may comprise a single compound or mixtures thereof with additional antibacterial agents. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or onto a site of infection, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

[0075] The compounds used in the method of the present invention can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

[0076] Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, 20 4894-7263-3313v.1Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

[0077] Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0078] The compounds used in the method of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue-targeted emulsions.

[0079] The compounds used in the method of the present invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl 21 4894-7263-3313v.1residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.

[0080] Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

[0081] For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non- effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.

[0082] Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. 22 4894-7263-3313v.1

[0083] The compounds used in the method of the present invention may also be administered via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.

[0084] Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

[0085] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

[0086] “And / or” as used herein, for example, with option A and / or option B, encompasses the separate embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.

[0087] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0088] Definitions: The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.

[0089] The term “subject” as used in this application means a mammal. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates including humans. Thus, the invention can be used in human medicine or also in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. 23 4894-7263-3313v.1The invention is particularly desirable for human medical applications. In a preferred embodiment the subject is a human.

[0090] The terms “therapeutically effective amount” or "amount effective to" encompasses, unless otherwise indicated, an amount sufficient to ameliorate or inhibit a symptom or sign of the medical condition. An effective amount for a particular subject may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects. An effective amount can be the maximal dose or dosing protocol that avoids significant side effects or toxic effects.

[0091] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0092] Purinergic hypothesis and immune system

[0093] G. Burnstock first depicted the term “purinergic” in 1972 due to the neurotransmitter action of adenosine triphosphate (ATP) being responsible for the unique transmission.

[0011] In 1976, he described the purinergic receptors located in cellular membrane, and in 1978, he identified the two different families named as P1 and P2 receptors due to their ligands adenosine and ATP / adenosine diphosphate (ADP) respectively.[12,13] In the early 1990’s P2 receptor subfamilies P2Y and P2X were recognized as P2Y which were found to be G-protein coupled receptors and ligand gated ion channel receptors respectively.[14,15] P2X receptors gained much of attention thanks to their selectivity towards ATP neurotransmitter and all other purine derivatives were found to be inactive.

[0016] Currently, seven P2X receptor subtypes are identified (P2X1-7) and among these subtypes P2X7 receptor (P2X7R) is reported to play a critical role in the development and progression of AD.

[0017] 24 4894-7263-3313v.1

[0094] In 2003, P2X7R activity was first linked to AD following the P2X7R overexpression observation in transgenic AD mouse brain.

[0018] In 2006, overexpression of P2X7R was reported in AD human brain isolated microglia compared to nondemented human brain isolated microglia and this action was linked to Aβ deposition.

[0019] Nowadays, P2X7Rs are known as key regulators in neuroinflammation. P2X7Rs are widely expressed in microglia and astrocytes while missing in neurons.

[0020] P2X7R mediated purinergic neurotransmission is activated with increased level of extracellular ATP which is released by either damaged neurons, microglia or astrocytes due to external particles such as Aβ oligomers and plaques in surrounding cells.[21–24]

[0095] In microglia, P2X7R activation induces the transition of the resting microglia to activated microglia through ERK and AKT pathways and mediate ATP-induced microglial cell death.

[0025] Activated microglia further generates several proinflammatory cytokines including IL- 1α, IL-1β, IL18,25 IL-6, TNF-α and chemokine CCL2

[0026] , among which IL-1β is one of the most widely studied cytokine. IL-1β is not produced by healthy resting microglia, it is mainly produced by activated microglia. IL-1β production is a multistep process and starts with the synthesis of immature pro-IL-1β via pathogen

[0027] or damage

[0028] associated molecular pattern triggered pattern recognition receptors (PRR) family activation. The inactive 31 kDa precursor pro-IL-1β further cleaved by the pro-inflammatory protease caspase-1 to generate bioactive 17 kDA sized IL-1β.

[0029] Caspase-1 activation is mediated by a multi-protein complex termed the inflammasomes which converts pro-caspase to caspase-1.

[0030] Among the characterized inflammasome pathways, cytosolic PRR triggered NLRP3 (nucleotide-binding, leucine-rich repeat, pyrin domain containing 3) is linked to neurodegenerative diseases.31 Although the secretion mechanism of mature IL-1β remains to be elucidated, P2X7R is considered to be one of the most potent drivers of NLRP3 inflammasome pathway activation and IL-1β maturation.

[0032] NLRP3 inflammasome activation is reported to play key role in AD pathology. NLRP3 inflammasome was identified as a sensor of Aβ in a process involving the phagocytosis of Aβ subsequently lysosomal damage and cathepsin B release. Furthermore, the NLRP3-IL-1β pathway was essential for the synthesis of proinflammatory and neurotoxic factors in microglia.

[0033] In another study, higher caspase-1 expression levels were observed in human mild cognitive impairment brain and AD brain samples. Furthermore, Nlrp3− / − or Casp1− / − mice models demonstrated protected memory loss, reduced caspase-1 and IL-1β activation, and increased Aβ 25 4894-7263-3313v.1clearance. In addition, NLRP3 inflammasome deficiency shifted microglial cells to M2 state and yielded decreased Aβ deposition in the APP / PS1 mice AD model.

[0034]

[0096] P2X7R was reported to be involved in Aβ mediated IL-1β release from activated microglia.[21,35] Furthermore, studies indicate pharmacological inhibition of P2X7R alleviated Aβ production through GSK3β and secretases in J20 mice familial AD model.[36,37] In another study, P2X7R deficiency reduced the Aβ lesions, rescued the cognitive impairment and enhanced the synaptic plasticity in APP / PS1 mice AD model. However, this activity was attributed to inhibition of Aβ-mediated CCL3 chemokine release rather than IL-1β.

[0038] NLRP3 inflammasome pathway also reported to mediate tau pathology as well. Recently, NLRP3 inflammasome deficiency is reported to alleviate tau hyperphosphorylation and aggregation through regulation of tau kinases and phosphatases in mice models. Moreover, Aβ injected tau transgenic mice models demonstrated that Aβ induces the tau pathology in an NLRP3-dependent manner.

[0039] In another study, pharmacological inhibition of P2X7R reported to yield reduced misfolded tau accumulation in hippocampal region and improved memory in transgenic P301S mice.

[0040] Similarly, pharmacological and genetic inhibition of P2X7R was also reported to reduce tau hyperphosphorylation in GSK3β dependent manner and rescues cognitive impairment in P301S mice.

[0041]

[0097] Apart from cytokines and chemokines, P2X7R also mediate reactive oxygen species (ROS) release from microglia through NADPH oxidase 2.

[0042] In astrocytes, although expressed to a lesser extent, P2X7R mediate NLRP2 inflammasome pathway together with pannexin-1 yielding caspase production and IL-1β release.

[0043] Again similarly, P2X7R also mediate ROS release from astrocytes though NADPH oxidase 2 and subsequently yield IL-6 production.

[0044] However, the contribution of astrocytic P2X7R to inflammation is much lesser compared to the cytokine / chemokine load generated by P2X7R mediated release from microglia. Therefore, astrocytic-P2X7R are attributed to be more likely to generate moderate signaling including; depressive-like behavior in rodents and major depression in humans through the interaction with cortico-limbic neural circuits.[45–47] Similarly, activation of astrocytic P2X7R are known to promote the release of gliotransmitters especially glutamate from astrocytes,[48,49] leading to glutamatergic (N-methyl-D-aspartate receptor) NMDAR activation and increased NMDAR- mediated astrocyte-neuron signaling.[49,50] The excessive and long-term glutamate release through P2X7R activation leads to excitotoxicity in neurons and oligodendrocytes.22,51,52 This 26 4894-7263-3313v.1effect was reported to be driven backwards and provide cytoprotective effect against ATP or Aβ42 by pharmacological inhibition of P2X7R.[22,51–53] Overall, the multi-effective nature of P2X7Rs upon various hypotheses made the search for P2X7R antagonist agents a trending approach in drug discovery studies for the treatment of AD.

[0098] Part I - Generation of novel compounds & In vitro results

[0099] We previously identified cholinesterase inhibition (ChEI)-based multi-target directed ligands (MTDLs) by building a library of seventy compounds having the scaffold of 4- methylbenzo[d]thiazole incorporated into piperazine derivatives with a propanamide linker. Compounds were initially screened for their in vitro acetyl / butyrylcholinesterase inhibition (AChEI / BuChEI), and twenty-two promising inhibitors were further characterized for their sigma 1 receptor (σ1R), sigma 2 receptor (σ2R), NMDAR-GluN2B binding affinities, and P2X7R antagonistic activities. Among tested compounds, five tertiary nitrogen bearing piperazine derivatives displayed a potent P2X7R antagonistic activity and we managed to build a structure- activity relationship (Figure 1A). Among them, 10 and 49 were selected for further studies due to their additional σ1R and NMDAR-GluN2B binding affinities respectively. Nine promising ChEI- based hit MTDLs were predicted to meet drug-likeness and ADME suitability for oral administration criteria in silico and displayed no in vitro cytotoxicity against human neuroblastoma (SH-SY5Y), human embryonic kidney (HEK293), human hepatoma (HepG2) cells. Compounds were then tested for their in vitro cytoprotective activities in two cell lines, SH-SY5Y and primary human astrocytes (pHAs), against the stress inducers H2O2and Aβ42, respectively. Seven potent cytoprotective compounds were screened for their in vivo efficacies using a zebrafish model of acute amyloidosis-induced synaptic degeneration. Adult zebrafish brains were treated with 20 μM Aβ42 either alone or in combination with seven hit ChEI-based MTDLs (10, 13−16, 19, and 49), donepezil at the concentration of 10 μM using cerebroventricular microinjection (CVMI) technique. Six compounds displayed equal / better synaptic protection than the benchmark drug donepezil. Among them, two P2X7R antagonist compound, 10 and 49, with IC50 values of 18.1 ± 2.08 μM and 1.13 ± 0.04 μM, respectively, were tested for their efficacies against Aβ42-induced inflammation. Immunostaining of L-Plastin (ortholog for human Lymphocyte Cytosolic Protein 1) antibody demonstrated that both P2X7R antagonists alleviated the activation state of microglia in vivo at five days after injection. Although compound 10 displayed a better anti-inflammatory profile, compound 49 was significantly more potent in rescuing the effect of Aβ42 in synaptic 27 4894-7263-3313v.1degeneration. This effect was attributed to the additional GluN2B modulator activity of 49 with a Ki value of 184 nM, indicating the beneficial effect of the MTDL approach. Since the compounds were tested using the CVMI route of administration, in vitro blood-brain barrier (BBB) and gastrointestinal tract (GIT) permeation assays were performed to assess successful membrane penetration. Among six hit ChEI-based MTDLs, compound 10 displayed an ambiguous penetration rate in both assays and was excluded from the hit series. The remaining five compounds were further screened for their CYP450 enzyme inhibition profile on three main subtypes 3A4, 2D6, and 2C9. Compound 49 strongly inhibited all three subtypes and was also excluded from the hit ChEI-based MTDLs series (Figure 1C). In the aspect of lead optimization studies, we aim to resolve the membrane penetration and CYP450 enzyme inhibition issues and enhance the P2X7R antagonistic activity by developing novel ChEI-based MTDLs.

[0100] We performed modifications on the benzo[d]thiazole aromatic ring in the context of lead optimization studies to with the aim of yielding enhanced binding efficacy towards tested targets, improved solubility and penetration rate. We introduced hydrogen bond acceptor methoxy groups at the sixth and fifth / sixth position to benzo[d]thiazole ring to increase structural similarity to donepezil (potent acetylcholinesterase (AChE) inhibitor and σ1R ligand) and ifenprodil (potent NMDAR-GluN2B ligand). Furthermore, we kept piperazine derivatives which previously yielded potent cholinesterase inhibition (Figure 2).

[0101] A general synthesis pathway for newly generated compounds is illustrated in FIG.3. The starting material 2 was commercially available. Compound 1 was synthesized by one-pot reaction of the 3,4-dimethoxyaniline with thiocyanogen generated from bromine and alkaline thiocyanate in glacial acetic acid medium in moderate yields.[54,55] Compounds 1 and 2 were then reacted with 3-chloropropionyl chloride in dichloromethane to yield N-acylated products 3 and 4 respectively.

[0056] As final step, N-alkylation was performed by reacting 3 and 4 with previously selected piperazine derivatives to yield target compounds.

[0056] The compounds were isolated as solid either by column chromatography using suitable solvent systems. Due to the presence of a piperazine moiety and an amide functional group, in the main scaffold, compounds are hygroscopic in nature. This property was previously confirmed in our single-crystal X-ray analysis.

[0056] Similar property was observed in our elemental analysis results. To enhance solubility, HCl salts of some compounds were obtained through the treatment of HCl(g) which 28 4894-7263-3313v.1was generated by drying HCl (35%) with H2SO4(98%) two times and transferred via a cannula to the balloon, containing compound in diethyl ether.

[0057]

[0102] σ1R, σ2R, and NMDAR-GluN2B Subunit Binding Affinity.

[0103] The σ1R, σ2R, and NMDAR-GluN2B radioligand binding affinities of newly generated and parent compounds was determined (data not shown). Introducing methoxy groups generally resulted in increased σ2R binding affinity and selectivity. This effect was most significantly observed with 4-pyridyl derivatives. The σ2R selective parent compound 49 has an SI of 5.65 and moderate binding affinity with a Ki value of 1700 nM.56 The binding affinity was increased with 6-methoxy substituted derivative 6-49 and 5,6-dimethoxy substituted derivative 5,6-49 with Ki and SI values of 563 nM 17.76 and 243 nM 41.15, respectively. In addition, NMDAR-GluN2B modulatory activity was maintained. In the 4-(t-butyl)benzyl derivative 63 analogs, 6-63 and 5,6-63, we observed increased NMDAR-GluN2B modulatory activity. Where the parent compound 63 did not display any significant NMDAR-GluN2B modulatory activity (Ki = >10000 nM), the methoxy-substituted analogs, on the other hand, showed five folds higher activity (6-63 Ki = 221 nM and 5,6-63 Ki= 218 nM).

[0104] In the aspect of lead optimization studies, we aimed to obtain MTDLs having a well- balanced affinity profile toward all targets of interest. Although GluN2B modulatory potency was preserved, the analogs of previously identified lead compounds, two potent and selective σ1R and σ2R ligands, the 2-tetrahydrofurylmethyl derivative 14 (σ1R Ki = 109 nM, SI= 45.87)56 and 2- (diisopropylamino)ethyl derivative 19 (σ2R Ki = 121 nM, SI= 6.68)56 respectively, failed to display such property. The σ1R binding affinity was drastically reduced with 6- methoxybenzo[d]thiazole derivative 6-14 (σ1R Ki = 615 nM) and 5,6-dimethoxybenzo[d]thiazole derivative 5,6-14 (σ1R Ki = 1800 nM). Despite the σ2R binding affinity in both analogs being slightly increased, affinity balance could not be achieved. Similarly, the σ1R binding affinity of 19 analogs was significantly reduced (Ki = >10000 nM). Although σ2R binding was slightly reduced, 6 / 5,6-methoxy substitution to benzo[d]thiazole resulted in even higher σ2R selectivity. While the σ2R SI of parent compound 19 was 6.68, the σ2R SIs of 6-19 and 5,6-19 were found to be 32.68 and 31.95, respectively. We were able to achieve the affinity balance with 15 and 16 analogs. We previously identified parent compound cyclohexyl derivate 15 as a potent and selective σ1R ligand (σ1R Ki = 24 ± 3 nM, SI = 9.7).56 The 6-methoxy substitution with 6-15 resulted in a slight decrease in σ1R binding affinity and increased σ2R affinity. Furthermore, 29 4894-7263-3313v.1NMDAR-GluN2B modulatory activity was increased and yielded the optimum balanced affinity towards all targets of interest (σ1R Ki = 48 ± 9 nM, σ2R Ki = 57 ± 18 nM, NMDAR-GluN2B Ki = 43 ± 2 nM). Introducing another methoxy group, 5,6-15, slightly reduced the binding potency towards all targets but maintained the affinity balance (σ1R Ki = 223 nM, σ2R Ki = 104 nM, NMDAR-GluN2B Ki = 112 nM). Another lead compound we identified, cyclohexylmethyl derivative 16, was a potent and selective σ1R ligand (σ1R Ki = 35 ± 9 nM, SI = 14.83) and potent NMDAR-GluN2B modulator (Ki = 60 ± 7 nM).56 Similarly, 6-16 and 5,6-16 analogs yielded improved affinity balance by a slight decrease in σ1R binding affinity, an increase in σ2R binding affinity, and maintaining the NMDAR-GluN2B modulatory activity. The butyl derivatives, 6-7 and 5,6-7, were another set of compounds displaying a well-balanced affinity profile. Therefore, we selected compounds 6-7, 5,6-7, 6-15, 5,6-15, and 6-16, 5,6-16 for further in vivo studies. Although hexyl derivative parent compound 13 analog 6-13 also displayed a considerable affinity balance, we did not include this compound in our test set. This was due to the lack of synaptoprotective efficacy against amyloidosis previously observed with the equipotent parent compound 13 in in vivo studies.

[0105] We also tested the analogs of previously identified P2X7R antagonists, compounds 10 and 49, for their P2X7R antagonistic activity. However, methoxy substitution on sixth, fifth and sixth positions on benzo[d]thiazole ring resulted in loss of activity.

[0106] In Vivo Synaptoprotective Activity of ChEI-Based MTDL Lead Analogs in Adult Zebrafish Model of Alzheimer’s Disease

[0107] We tested the neuroprotective effect of the selected new hit ChEI-based MTDLs in an adult zebrafish amyloidosis model, demonstrating AD-like symptoms, including synaptic degeneration and blood-brain barrier modulation.58–61 Adult zebrafish brains were treated with 20 μM Aβ42 either alone or in combination with new hit MTDLs (6-7, 5,6-7, 6-15, 5,6-15, and 6- 16, 5,6-16), and donepezil at the concentration of 10 μM using cerebroventricular microinjection (CVMI) technique. After five days post-injection, immunostaining was performed on telencephalon sections for a synaptic marker using synaptic vesicle glycoprotein 2 (SV2) antibody (Figure 4(A)-6(A)). Aβ42 reduced the synaptic density by 22.19 ± 1.28% five days after injection compared to the vehicle-injected control group. The synaptoprotective activities of butyl derivative 7, its analogs 6-7, and 5,6-7, and donepezil were shown. Introducing methoxy groups gradually yielded higher synaptoprotective activity compared to the Aβ42 control group. Treatment with 30 4894-7263-3313v.1parent compound 7 yielded a rescue effect of 16.09 ± 7.33%, whereas 6-7 and 5,6-7 demonstrated rescue effects of 23.17 ± 3.81% and 32.23 ± 3.91%, respectively. Furthermore, parent compound 7 did not show any superior activity to donepezil (p = <0.05). An increase in σ2R binding affinity yielded a better synaptic protective profile. The 6-methoxybenzo[d]thiazole derivative 6-7 demonstrated 10.60 ± 1.67% higher efficacy than donepezil (p = 0.034). Interestingly, despite significantly reduced binding affinity towards all targets of interest, treatment with 5,6-7 yielded an even higher 19.67 ± 3.21% synaptic density than donepezil (p = <0.0001). Furthermore, 5,6-7 treatment displayed 9.79 ± 2.87% higher synaptic density than control levels (p = 0.0347).

[0108] Lead optimization studies on cyclohexyl derivative 15 did not yield enhanced potency in our in vivo model. Despite being the most potent σ1R, σ2R ligand, and NMDAR-GluN2B modulator, the 6-methoxybenzo[d]thiazole derivative 6-15 did not demonstrate any statistically significant difference in synaptic density in vivo compared to the parent compound 15. Similarly, with a slight reduction in potency towards all targets of interest, the 5,6-methoxybenzo[d]thiazole derivative 5,6-15 did not yield any change in synaptoprotective activity. However, the binding affinity difference was not drastic as the butyl analogs. Thereby, it might not produce any significant effect in vivo. Nevertheless, compounds 6-15 and 5,6-15 displayed equipotency to 15 and donepezil and significantly rescued the effect of Aβ42.

[0109] The synaptoprotective activities cyclohexylmethyl derivatives 6-16, 5,6-16 and their parent compound 16 are shown in Figure 5. Similarly with the cyclohexyl derivatives, we did not observe any significant difference in synaptoprotective potency with the novel analogs compared to parent compound 16. This activity again could be due to slight change in binding affinities among the derivatives. Nonetheless, treatment with both analogs 6-16 and 5,6-16 yielded better synaptic integrity protection than benchmark drug donepezil by 15.27 ± 3.19% (p = 0.0038) and 13.55 ± 2.31% (p = 0.0332), respectively. Treatment with parent compound 16 rescued the effect of Aβ42 by 12.01 ± 7.15% higher than donepezil (p = 0.0229).

[0110] In Vitro Pharmacokinetics Profiling, Permeability Assessment, GIT and BBB Permeation.

[0111] We performed barrier permeation studies for the newly generated hit compounds and their parent lead compounds using a parallel artificial membrane permeability assay (PAMPA). The blood-brain barrier (BBB) and gastrointestinal (GIT) passive diffusion penetration assessment was done at the pH values of 7.4 and 6.8, respectively. The efficient permeability rates (Pe) of all 31 4894-7263-3313v.1reference drugs were similar to the previously published data in both assays.[62,63] As expected, the substitution of hydrogen bond acceptor methoxy groups yielded increased solubility and maintained lipophilic character. Hence, the penetration rates of newly generated analogs were higher than the corresponding parent compounds. Due to their high BBB-PAMPA penetration rates, we selected compounds 6-15, 5,6-15, 6-16, 5,6-16, and 6-7 for further 3D cell culture studies.

[0112] Dose determination for starPEG-Heparin hydrogel-based 3D cultures of human fetal primary astrocytes.

[0113] To determine the safe doses of newly generated lead ChEI-based MTDLs for further cytoprotective activity assay on the 3D cell culture AD model, we tested the compounds 6-7, 6- 15, 5,6-15, 6-16, and 5,6-16 at the concentrations of 1 μM, 5 μM, 1 μM, 5 μM, 5 μM, respectively. The highest safe doses were selected based on the previously performed 2D cytotoxicity assay on primary human astrocytes (pHAs). The pHA-encapsulated starPEG-Heparin hydrogels were treated with newly generated lead ChEI-based MTDLs at the respective concentrations for 10 weeks. After treatment every two days for 10 weeks, hydrogels were fixed, and immunocytochemical staining was performed for acetylated tubulin and glial fibrillary acidic protein (GFAP) (Figure 7(A)). The safety assessment was performed by determining acetylated tubulin and GFAP-positive cell volume. As shown in Figure 7(B)-7(D), compounds did not affect the development and differentiation of astrocytes to neurons after 10 weeks of incubation.

[0114] The cytoprotective activity assessment on AD-like model of starPEG-Heparin hydrogel-based 3D pHAs.

[0115] We tested the cytoprotective activity of newly generated lead ChEI-based MTDLs on Aβ42 treated 3D pHA hydrogels that serves a model for human AD pathology.[64,65] Aβ42 treated pHAs were encapsulated with starPEG-heparin hydrogels and further treated with compounds 6-7, 6-15, 6-16, 5,6-15, and 5,6-16 every two days at the designated concentrations for 10 weeks. The cytoprotective activity assessment was performed by determination of acetylated tubulin- and GFAP-positive cell filament lengths (Figure 8(B)-8(C)).

[0116] Methods

[0117] Chemistry

[0118] The general reaction pathway is illustrated in Fig. 3. Novel compounds with the scaffold shown in the upper panel are aimed to be synthesized in three steps. For step 1, the Strecker reaction is aimed to be performed by reacting cyclic ketones with secondary amine 32 4894-7263-3313v.1piperazine derivatives in the presence of potassium cyanide (KCN) according to published protocols[68,75]. To be obtained, nitrile intermediates will then be reduced to primary amines using lithium aluminum hydride (LiAlH4) to yield the general structure A75. In step 2, aromatic carboxylic acids will be converted to acyl chlorides using either oxalyl chloride

[0068] or thionyl chloride (SOCl2)

[0078] in dichloromethane (DCM): dimethylformamide (DMF) (9:1) to yield the general structure B. In step 3, N-acylation will be performed by reacting the general structures of A and B under basic conditions using either potassium carbonate (K2CO3) in acetonitrile (ACN)75 or N,N-Diisopropylethylamine (DIEPA) in dichloroethane (DCE)

[0075] to generate the targeted final products having the general structure of C. Obtained novel compounds will be characterized using spectroscopic methods; 1H-NMR, 13C-NMR, FT-IR, LC-MS and their impurities will be determined using either elemental analysis or HRMS.

[0119] Preparation of 5,6-dimethoxybenzo[d]thiazol-2-amine (A; CAS number: 6294-52-6)

[0120] 32.64 mmol (5 g) of 3,4-dimethoxyaniline and equimolar potassium thiocyanate (32.64 mmol, 3.172 g) were dissolved in glacial acetic acid (15 mL), The temperature was cooled to 0−5 °C, then a solution of bromine (43.52 mmol, 2.23 mL) in acetic acid (10 mL) was added dropwise 3-(4-butylpiperazin-1-yl)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)propenamide to the reaction medium. After the dropping process was completed, the reaction contents were stirred at room temperature for 12 hours. TLC controls were performed using the mobile phase system of dichloromethane: methanol: triethylamine (90:9:1). The reaction contents were poured into iced- water and then pH was adjusted to 10 by adding dropwise of 1N sodium hydroxide solution. The lilac colored precipitate was filtered, dried and recrystallized from ethanol.[1,2]

[0121] Reagents:

[0122] 1) IUPAC name: 3,4-Dimethoxyaniline CAS number: 6294-52-6 Supplier: Sigma Aldrich Catalog number: A83008

[0123] 2) IUPAC name: Potassium thiocyanate CAS number: 333-20-0 Supplier: Sigma Aldrich Catalog number: 207799

[0124] 3) IUPAC name: Glacial acetic acid 33 4894-7263-3313v.1CAS number: 64-19-7 Supplier: Sigma Aldrich Catalog number: 100063

[0125] 4) Bromine CAS number: 7726-95-6 Supplier: Sigma Aldrich Catalog number: 470864

[0126] 5) Sodium hydroxide CAS number: 1310-73-2 Supplier: Sigma Aldrich Catalog number: 221465 Solvents:

[0127] 1) Dichloromethane CAS number: 75-09-2 Supplier: Sigma Aldrich Catalog number: 34856

[0128] 2) Methanol CAS number: 67-56-1 Supplier: Sigma Aldrich Catalog number: 34860

[0129] 3) Triethylamine CAS number: 121-44-8 Supplier: Sigma Aldrich Catalog number: T0886

[0130] Preparation of 3-chloro-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)propanamide (B) 2-Amino-6-methoxy / 5,6-dimethoxybenzo[d]thiazole (20 mmol, 3.38 g) was dissolved in tetrahydrofuran (THF). The temperature was cooled to 0−5 °C, then 33 mmol (3.21 mL) of 3- chloropropionyl chloride was then added to the solution dropwise. After being stirred for 1 h, the reaction mixture was allowed to warm to room temperature and stirred for 36 h. TLC controls were performed using the mobile phase system of benzene / methanol (9:1). 34 4894-7263-3313v.1

[0131] Precipitated crude product was then filtered, washed with THF, and dried. THF in the filtrate was evaporated under reduced pressure. Both remaining solids were first washed with 5% NaHCO3 solution and then several times with distilled water. Obtained solids were then recrystallized from ethanol.[3]

[0132] Reagents

[0133] 1) IUPAC name: 3-chloropropionyl chloride CAS number: 625-36-5 Supplier: Sigma Aldrich Catalog number: C69128

[0134] 2) Sodium bicarbonate CAS number: 144-55-8 Supplier: Sigma Aldrich Catalog number: S6014

[0135] Solvents:

[0136] 1) Tetrahydrofuran CAS number: 109-99-9 Supplier: Sigma Aldrich Catalog number: 401757

[0137] 2) Benzene CAS number: 71-43-2 Supplier: Sigma Aldrich Catalog number: 401765

[0138] 3) Ethyl acetate CAS number: 141-78-6 Supplier: Sigma Aldrich Catalog number: 319902

[0139] 4) Hexane CAS number: 110-54-3 Supplier: Sigma Aldrich Catalog number: 296090 35 4894-7263-3313v.1

[0140] Preparation of N-(6-methoxy / 5,6-dimethoxybenzo[d]thiazol-2-yl)-3-(4- substitutedpiperazin-1-yl)propenamide 1.29 mmol (0.35 g) and 1.16 mmol (0.35 g) of 3-chloro- N-(5,6-dimethoxybenzo[d]thiazol-2-yl)propanamide and equimolar piperazine derivatives were dissolved in acetone (20 mL), to this solution equimolar anhydrous potassium carbonate was added and stirred for 12 hours at room temperature. TLC controls were performed using the mobile phase system of benzene: methanol (9:1). Precipitates were removed by filtration and acetone was evaporated under reduced pressure. Remaining crude products were then purified by either recrystallized from appropriate solvents or column chromatography using appropriate mobile phase systems.[3]

[0141] Reagents:

[0142] 1) IUPAC name: 1-Butylpiperazine CAS number: 5610-49-1 Supplier: Enamine Catalog number: EN300-256519

[0143] 2) IUPAC name: 1-Cyclohexylpiperazine CAS number: 17766-28-8 Supplier: Enamine Catalog number: EN300-110637

[0144] 3) IUPAC name: 1-(Cyclohexylmethyl)piperazine CAS number: 57184-23-3 Supplier: Enamine Catalog number: EN300-58708

[0145] 4) Potassium carbonate CAS number: 584-08-7 Catalog number: 209619

[0146] Solvents:

[0147] 1) Acetone CAS number: 67-64-1 Supplier: Sigma Aldrich Catalog number: 179124

[0148] 2) Ethanol 36 4894-7263-3313v.1CAS number: 64-17-5 Supplier: Sigma Aldrich Catalog number: 100986

[0149] 3) Diethyl ether CAS number: 60-29-7 Supplier: Sigma Aldrich Catalog number: 296082

[0150] Part II - In vivo results

[0151] Results

[0152] MTDL treatment significantly improved cognitive performance in APP-KI mice, as evidenced by their performance in the T-maze and NOL tests. In the T-maze, MTDL-treated mice spent significantly more time in the acquisition arm, indicating improved memory compared to APP-KI vehicle-treated mice (see Figure 12). This effect was quantified in panel 14B, showing significant improvement in both the time spent in the novel arm and the T4 / T5 ratio. Similarly, in the NOL test, MTDL-treated mice demonstrated a higher preference for the novel object location, which was significantly reflected in the discrimination index (DI) quantification. Furthermore, synaptic integrity, as assessed by synaptophysin staining, was notably compromised in APP-KI mice, with a reduction in both the surface area and number of synaptic puncta. MTDL treatment restored these parameters to levels similar to those observed in control mice, suggesting that MTDL has a protective effect against synaptic degeneration in this model.

[0153] Methodology

[0154] Mouse model: The APP-NL-G-F knock-in mouse model, which harbors the humanized amyloid precursor protein (APP) gene with Swedish (KM670 / 671NL), Iberian (I716F), and Arctic (E693G) mutations, was used in this study. These mice were chosen because they develop amyloid plaques, synaptic loss, and cognitive deficits similar to those observed in human Alzheimer’s disease (AD). The APP-NL-G-F model is known for its accelerated pathology, making it a suitable model for studying the effects of therapeutic interventions on AD-like symptoms.

[0155] Maintenance: All mice were housed under specific pathogen-free conditions with controlled temperature and a 12-hour light / dark cycle. They had ad libitum access to food and water. 37 4894-7263-3313v.1

[0156] Ethics: All animal procedures were performed in accordance with institutional guidelines and approved by Columbia IACUC.

[0157] Age and sex: Male and female APP-NL-G-F mice were used in the study, aged between 6 and 8 months at the start of the experiments. This age range was selected because the mice exhibit robust amyloid pathology and cognitive deficits by this stage.

[0158] Drug administration: Multi-target directed ligand (MTDL) was administered to the APP-NL-G-F mice. The treatment was delivered via osmotic mini-pumps (Alzet model 1002) implanted subcutaneously. The mini-pumps were connected to a cannula placed into the lateral ventricle for continuous intracerebroventricular (ICV) infusion.

[0159] Concentration and duration: MTDL was delivered at a concentration of 2 μM over a 14-day period. Control groups received vehicle infusion under the same conditions.

[0160] Surgical procedure: Mice were anesthetized using isoflurane (2-3% for induction and 1.5% for maintenance) and placed in a stereotaxic frame. The cannula was implanted at coordinates relative to bregma (AP: -0.2 mm, ML: +1.0 mm, DV: -2.0 mm) and secured with dental cement. The mini-pumps were placed subcutaneously in the dorsal region.

[0161] Post-surgical care: Post-operative care included administration of analgesics (e.g., buprenorphine) and monitoring for any signs of distress or infection.

[0162] Mouse novel object location (NOL) test: Mice were habituated to the testing arena for two days before testing. On the test day, two identical objects were placed in the arena, and mice were allowed to explore for 5 minutes. After a retention interval of 1 hour, one object was moved to a novel location, and mice were returned to the arena for a 5-minute test session. Time spent exploring each object was recorded, and a discrimination index (DI) was calculated as the ratio of the time spent exploring the novel object to the total time spent exploring both objects.

[0163] T-Maze test: The T-maze consists of three arms: one start arm and two goal arms (retention and acquisition). Mice were placed in the start arm and allowed to explore the maze. The time spent in each arm was recorded. The test was conducted in two phases: an acquisition phase where the mouse explores one arm and a test phase after a delay where the mouse is introduced back into the maze, and the time spent in each arm is recorded. The T4 / T5 ratio (time spent in the novel arm at time 5 over time 4) was calculated to assess memory retention. 38 4894-7263-3313v.1

[0164] Tissue preparation: Following the treatment period, mice were sacrificed by cervical dislocation, perfused with saline followed by 4% paraformaldehyde, and the brains were collected for further analysis, including sectioning and immunostaining.

[0165] Image analyses: The images acquired from the confocal microscope were imported into Fiji ImageJ. The images were typically in .czi format (from Zeiss Zen) and converted to compatible formats such as .tiff for ease of analysis. This conversion process ensured that the images retained their high resolution and detail, necessary for accurate 3D reconstruction and subsequent analysis. Using Fiji ImageJ’s 3D Viewer plugin, the imported images were reconstructed into an object. This reconstruction provided a more accurate and comprehensive representation of synaptophysin- positive puncta distributed throughout the brain section, allowing for spatial analysis within the tissue. The thresholding process was a critical step in distinguishing synaptophysin-positive puncta from the background noise in the images. The threshold level was carefully selected through an iterative process. An initial threshold value was applied based on the intensity histogram of the images. This value was chosen automatically to ensure that most of the puncta were included while minimizing the inclusion of background noise. The threshold was then manually adjusted by visually inspecting multiple sections within the image. The goal was to exclude non-specific background fluorescence while retaining true synaptic puncta. This process involved comparing the binary mask created by the thresholding with the original image to confirm that the puncta detected were biologically relevant. The selected threshold was applied uniformly across all images to ensure consistency in the analysis. Any variations in staining intensity between samples were accounted for by adjusting the threshold so that the relative brightness of synaptophysin- positive areas remained comparable across different sections. This careful selection and consistent application of the threshold ensured that only relevant synaptic structures were included in the analysis, reducing the likelihood of artifacts affecting the quantification. After thresholding, the 3D Objects Counter plugin in Fiji ImageJ was used to identify and quantify individual puncta within the 3D volume. This plugin detects objects based on user-defined parameters such as size, shape, and intensity, which were set to exclude any remaining noise and to focus on true synaptic structures. The plugin provided a comprehensive count of the number of puncta, their total volume, and their spatial distribution within the brain section. The data generated by the 3D Objects Counter was exported as a detailed dataset, including measurements such as the number of puncta, their individual volumes, and their distribution patterns. This dataset was then analyzed 39 4894-7263-3313v.1statistically to assess differences in synaptic density and morphology across different experimental conditions, providing insights into the effects of the treatments on synaptic integrity. One way ANOVA with Tukey’s post hoc test was used for quantification analyses. Data analysis was performed using GraphPad Prism, a software package widely used for statistical analysis in biological research. * (p < 0.05), ** (p < 0.01), *** (p < 0.001), ns / nd (not significant, p > 0.05).

[0166] Conclusion

[0167] Administering a multi-target directed ligand (MTDL) to APP-NL-G-F mice via intracerebroventricular (ICV) infusion using osmotic mini-pumps at a concentration of 2 μM over a 14-day period results in a significant increase in time spent in the acquisition arm of a T-maze test compared to vehicle-treated control mice. Multi-target directed ligand (MTDL) administered at a concentration sufficient to improve cognitive performance and restore synaptic integrity in APP-NL-G-F mice.

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Claims

Claims 1. A compound represented by the formula ‒CH(CH3)2, ‒C(CH3)3, (CH3)2, 2-tetrahydrofurylmethyl,cyclohexyl, cyclohexylmethyl, ‒CH2CH2N(CH(CH3)2)2, or 2-pyridyl, 4-pyridyl, or 2- pyrimidyl; and wherein: Ar is ,Ar is , wherein R is ‒H, ‒Cl, ‒OCH3, or ‒CF3; or51 4894-7263-3313v.1, o hereof.

2. The compound of claim 1, represented by the formula wherein:R1 is ‒H or ‒OCH3, and R is ‒CH2(CH2)2CH3, ‒CH2(CH2)4CH3, cyclohexyl, or cyclohexylmethyl; or R1 is ‒OCH3 and R is 4-t-butylbenzyl, or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 2, represented by the formula or a4. The compound according to claim 2, represented by the formulaor a 52 4894-7263-3313v.

15. The compound according to claim 2, represented by the formula or a6. A pharmaceutical composition comprising: the compound according to any one of claims 1-5; and an excipient.

7. A method for treating a neurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of the compound according to any one of claims 1-5 or an effective amount of the pharmaceutical composition according to claim 6.

8. The method of Claim 7, wherein treating the disease comprises improving one or more cognitive performance measures.

9. A method for preventing neurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of the compound according to any one of claims 1-5 or an effective amount of the pharmaceutical composition according to claim 6.

10. A method of improving synaptic integrity in a subject having an impaired synaptic integrity associated with a neurodegenerative disease, the method comprising the step of administering to a subject in need thereof an effective amount of the compound according to any one of claims 1-5 or an effective amount of the pharmaceutical composition according to claim 6. 53 4894-7263-3313v.

111. The method of Claim 10, wherein administering the effective amount of the compound or composition effects an increase in surface area of and / or number of synaptic puncta.

12. The method of any of Claims 7 - 11, wherein the neurodegenerative disease is Alzheimer’s disease.

13. A method for producing the compound according to any one of claims 2-5, comprising the steps of: reacting with thiocyanogen to ;reacting with 3-chloropropionyl chloride to obtainreacting to obtain.

14. A method for producing a compound represented by the formula wherein:54 4894-7263-3313v.1R is , wherein R’ is ‒CH3, ‒CH(CH3)2, ‒C(CH3)3, ‒CH2(CH2)2CH3, CH2CH2CH2N(CH3)2, 2-tetrahydrofurylmethyl, cyclohexyl, cyclohexylmethyl, ‒CH2CH2N(CH(CH3)2)2, 2-pyridyl, 4-pyridyl, or 2-pyrimidyl; and wherein: ,the method comprising the steps of:reacting Ar‒NH2 with 3-chloropropionyl chloride to obtain Ar‒NH‒CO‒(CH2)2‒Cl; and to .

15. The method of claim 13 or 14, wherein thiocyanogen is generated from bromine and alkaline thiocyanate. 55 4894-7263-3313v.1

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