Pyrazolone compounds for use in treatment of neurodegenerative disease

Abstract

Pyrazolone compounds, pharmaceutical compositions comprising the same, and methods of using the same are disclosed herein. The disclosed compounds may be used for treatment of neurodegenerative diseases and / or disorders, such as Alzheimer's disease.

Description

[0001] PYRAZOLONE COMPOUNDS FOR USE IN TREATMENT OF NEURODEGENERATIVE DISEASE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U. S. Provisional Patent Application No. 63 / 715,322 filed November 1, 2024. The content of which is hereby incorporated by reference in its entirety.

[0003] STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] This invention was made with government support under grant number AG061708 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] BACKGROUND OF THE INVENTION

[0006] Presently more than 50 million people, mostly in an advanced age, are affected by dementia, and, with the increasing life expectancy of the worldwide population, the number is expected to exceed 150 million by 2050. Alzheimer's disease (AD) is the most common cause of dementia and one of the largest public health challenges of this century.2Throughout the disease, the patients experience a continuous loss of cognitive functions, neurobehavioral changes, and functional decline, eventually leading to a complete loss of independence, severely impacting the patients and their families' daily lives.2-3The currently approved therapies for AD, the acetylcholinesterase inhibitors (donepezil, galantamine, and rivastigmine), NMDA antagonist memantine, antipsychotic brexpiprazole, and antiamyloid antibodies lecanemab and donanemab, which have numerous side effects, are only capable of providing minimal symptomatic relief to the patients.4-5Hence, with the continuous increase of AD prevalence there is an urgent need for novel and more efficient therapies that could decrease the social and economic tolls on the patients, their families, and the global healthcare system.6

[0007] AD is a neurological disorder characterized by abnormal processing and polymerization of soluble proteins that lead to the formation of protein aggregates that contribute to the progressive decline observed throughout the disease.2Indeed, amyloid (A0) plaques and neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein are established hallmarks of AD and are used as markers of the disease for diagnosis.7The A0 peptide plaque formation precedes by two to three decades the onset of AD and plays a crucial role in the disruption of the neuronal networks and synaptic loss that leads to the disease progression. Additionally, a key correlation between AP accumulation and hyperphosphorylated tau NFTs formation in patients’ brains has also been reported.8Hence, due to its role in AD, A has been a key target for the development of novel, disease-modifying therapies.2, 9

[0008] In the last decade, several therapeutic approaches targeting different stages of Ap accumulation were developed and evaluated in clinical trials.1, 9-10One approach was based on the use of monoclonal antibodies (mAb) designed to target monomeric or oligomeric forms of Ap and clear accumulated plaques in the patients brains.9The recent FDA approval of aducanumab partially validated the potential of the Ap targeted therapies for the development of disease-modifying drugs.9However, despite the promising results, brain haemorrhages, vasogenic edemas, and brain volume loss, among other side effects, were observed during clinical trials with Ap mAbs.1Indeed, based on similar observations soon after aducanamab's approval, the FDA limited its use to patients with mild AD or cognitive impairment.1Furthermore, the relatively low blood-brain barrier (BBB) penetration and the need for medical centers specially prepared for Ap mAbs administration significantly limits the use of these therapies.9Therefore, novel treatments with better efficacy and safety profiles that would be available to a much broader range of patients are needed. Additionally, a wealth of scientific evidence has demonstrated the multifactorial character of AD.11Hence, the development of small molecules capable of permeating the BBB and designed to synergistically target Ap and modulate the activity of other key biological targets behind AD pathological processes may provide therapies that more efficiently meet the patients’ needs.11

[0009] BRIEF SUMMARY OF THE INVENTION

[0010] Disclosed herein are pyrazolone compounds and methods of using pyrazolone compounds in treatment of Alzheimer’s disease (AD).

[0011] One aspect of the present disclosure provides a method of treating Alzheimer’s disease in a subject in need thereof. The method comprises administering to the subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof; or a pharmaceutical composition comprising the compound of formula (I) and a pharmaceutically acceptable excipient, carrier, or diluent;

[0012]

[0013] wherein

[0014] R1is H or methylene;

[0015] R2is halogen;

[0016] x is 0-4;

[0017] L is -O-(CH2)n- -CH2-N+HRA-CH2- -(CH2)n-,-N(RA)-, or -N(RA)-(CH2)n-; RAis C1-6alkyl; or RAtogether with R1form (CH2)m;

[0018] n is 0-3;

[0019] m is 1-2;

[0020] M is a counterion; and

[0021] y is 0 or 1.

[0022] Another aspect of the present disclosure provides a method of inhibiting amyloid-P aggregation and / or reducing amyloid-P aggregation-induced toxicity. The method comprises contacting amyloid P with the compound disclosed herein.

[0023] Another aspect of the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof:

[0024]

[0025] wherein

[0026] R1is H or methylene;

[0027] R2is halogen;

[0028] x is 0-2;

[0029] L is — (CH2)n—,— N(RA)—, or -N(RB)-(CH2)n-;

[0030] RAis Ci-salkyl;

[0031] RBtogether with R1form -CH2-;

[0032] n is 0-2

[0033] M is a counterion; y is 0 or 1; and

[0034] with the proviso that

[0035] when L is -N(RB)-(CH2)n- and n is 2, R2is not -3,5-dichloro.

[0036] Another aspect of the present disclosure provides a pharmaceutical composition, comprising the compound disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0039] Figure 1 shows the chemical structures of edaravone and the arylpyrazolone compounds evaluated for their protective activity against A-P induced toxicity on MC65 neuronal cells.

[0040] Figure 2 shows the enol forms observed in edaravone and on the pyrazolone compound series responsible for the radical scavenging activity.

[0041] Figure 3 shows the time-dependent concentration profiles of orally administered 1 in mouse blood and brain.

[0042] Figure 4 shows the dose-dependent curves of aryl pyrazolones 1-4 and edaravone on A intracellular aggregation toxicity studies on MC65 cells.

[0043] Figure 5 shows the dose-dependent curves of aryl pyrazolones 1-4, 5, 7 in anti-ferroptotic toxicity studies on HT22 cells treated with RSL3.

[0044] Figure 6 shows the dose-dependent curves of aryl pyrazolones 1 and 2 in anti-ferroptotic toxicity studies on HT22 cells treated with glutamate.

[0045] Figure 7 shows the time-dependent concentration profiles of iv administered 1 in mouse blood and brain.

[0046] Figure 8 shows the HPLC chromatogram of compound 3.

[0047] Figure 9 shows the HPLC chromatogram of compound 4 (solubilized from DMSO solution - solv. peak 0.32 min.)

[0048] Figure 10 shows the HPLC chromatogram of compound 5. Figure 11 shows the HPLC chromatogram of compound 6.

[0049] Figure 12 shows the HPLC chromatogram of compound 7.

[0050] DETAILED DESCRIPTION OF THE INVENTION

[0051] The present disclosure relates to pyrazolone compounds and methods of using pyrazolone compounds in treatment of neurodegenerative diseases and / or disorders, such as Alzheimer’s disease (AD). AD incurs heavy costs for both the population and health systems. Nevertheless, drugs available only provide minimal symptomatic management without much impact on the patients’ quality of life. The multifactorial character of AD suggests that the development of new therapies modulating multiple biological targets contributing to disease progression will be more effective treating the disease. Therapies targeting amyloid-beta oligomers may be a valid approach for the development of more efficacious therapies for AD. Oxidative stress has been associated with the progression of AD. The design and evaluation of a series of pyrazolone compounds for their activity against A aggregation toxicity and oxidative stress are disclosed herein.

[0052] Chemical Entities

[0053] The term "alkyl" refers to a straight-chain, branched, or cyclized alkyl radical in all its isomeric forms, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as Ci-Ci2-alkyl, Ci-Cio-alkyL and Ci-Cs-alkyl, respectively.

[0054] The term "alkylene" refers to a diradical of straight-chain or branched alkyl group (e.g., a diradical of straight-chain or branched C1-C12 alkyl group). Exemplary7alkydene groups include, but are not limited to -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)CH2-, -CH(CH2CH3)CH2-, and the like.

[0055] The term "alkenyl" refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C 12- alkeny l (or C2-i2alkenyl), C2-Cio-alkenyl (or C2-walkenyl), and C2-Ce-alkenyl (or C2-6alkenyl), respectively.

[0056] The terms "alkoxy" or "alkoxyl" refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, tertbutoxy and the like.

[0057] The term "cycloalkyl" refers to a monovalent saturated or partially saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8. or 4-6 carbons, referred to herein, e.g., as "C4-8-cycloalkyl," derived from a cycloalkane. Unless specified otherwise, the cycloalkyl group is not substituted, i.e., it is unsubstituted. The term "heterocycloalky l" (or "heterocyclyl") refers to a monovalent saturated or partially saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8. or 4-6 carbons in which at least one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and / or S.

[0058] The term “halo” or “halogen” refers to a halogen atom or halogen radical (e.g., -F, -Cl, -Br, or -I).

[0059] The term "haloalkyl" refers to an alkyl group that is substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, and the like.

[0060] The term "aryl" refers to a carbocyclic aromatic group. The term "aryl" includes monocyclic ring systems, and polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. Unless specified otherwise, the aryl ring is unsubstituted. In certain embodiments, the ary l group is a 6-10 membered ring structure. Representative aryl groups include phenyl, naphthyl, anthracenyl, 1.3-benzodioxolyl and the like.

[0061] The substituents on the aryl (e.g., phenyl) rings in the compounds of the disclosure may be at ortho-, meta-, or para- positions.

[0062] The term "heteroaryl" refers to an aromatic 5- to 10-membered ring structure, alternatively 6- to 10-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heteroaryl group can be specified using Cx-Cx nomenclature where x is an integer specifying the number of ring atoms. For example, a C3-C7 heteroaryl group refers to an aromatic 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The term "heteroaryl" includes monocyclic ring systems, and polycyclic ring systems having two or more heterocyclic rings in which two or more carbon or heteroatom are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is a heterocyclic aromatic group and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. Representative heteroaryl groups include pyridinyl, quinolinyl, furanyl, thionyl, indolyl, and the like.

[0063] The term “optionally substituted” refers to zero, one or more carbon atoms in the group being independently substituted with one or more functional groups described herein. The compounds of the disclosure may contain one or more chiral centers and / or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term "stereoisomers" when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols "R" or "5," or "+" or depending on the configuration of substituents around the stereogenic carbon atom and or the optical rotation observed. The present invention encompasses various stereo isomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated (±)" in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise. Also contemplated herein are compositions comprising, consisting essentially of, or consisting of an enantiopure compound, which composition may comprise, consist essentially of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%. or 100% of a single enantiomer of a given compound (e.g., at least about 99% of an R enantiomer of a given compound).

[0064] As used herein, "salt" refers to acid addition salts and basic addition salts. It may also refer to those salts that may be prepared in situ during the final isolation and purification of the compounds of the invention.

[0065] Examples of acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isothionate), lactate, malate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitate, pectinate, persulfate, 3 -phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesul fonate and undecanoate. Also, the basic nitrogen-containing groups may be quatemized with such agents as lower alky l halides such as, but not limited to, methyl, ethyl, propyl, and buty l chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as, but not limited to, decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzy l and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid and such organic acids as acetic acid, fumaric acid, maleic acid, 4-methylbenzenesulfonic acid, succinic acid, and citric acid.

[0066] Basic addition salts may be prepared in situ during the final isolation and purification of compounds of this invention by reacting a carboxylic acid-containing moiety with a suitable base such as, but not limited to, the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary’ or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as, but not limited to, lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary’ ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like. Other examples of organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.

[0067] Compounds described herein may exist in unsolvated as well as solvated forms, including hydrated forms, such as hemi-hydrates. In general, the solvated forms, with pharmaceutically acceptable solvents such as water and ethanol among others are equivalent to the unsolvated forms for the purposes of the invention.

[0068] Methods

[0069] One aspect of the present disclosure provides a method of treating neurodegenerative diseases and / or disorders, such as Alzheimer’s disease in a subject in need thereof. The method comprises administering to the subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof; or a pharmaceutical composition comprising the compound of formula (I) and a pharmaceutically acceptable excipient, carrier, or diluent;

[0070]

[0071] wherein

[0072] R1is H or methylene;

[0073] R2is halogen;

[0074] x is 0-4; L is —O-(CH2)n—, -CH2-N+HRA-CH2- -(CH2)n-,-N(RA)-, or -N(RA)-(CH2)n-; RAis C1-6alkyl; or RAtogether with R1form -(CH2)m-;

[0075] n is 0-3;

[0076] m is 1-2;

[0077] M is a counterion; and

[0078] y is 0 or 1.

[0079] In some embodiments, L is -O-CH2- In some embodiments, L is -CH2-N HMe-CH2- In some such embodiments, M is H2PO4‘ and y is 1.

[0080] In some embodiments, L is -(CH2)n- and n is 0 or 2.

[0081] In some embodiments, L is -NMe- In some embodiments, R1is H.

[0082] In some embodiments, L is -N(RA)-CH2-, and RAtogether with R1form -Chhin some embodiments, R2is -Cl and x is 2.

[0083] In some embodiments, R2is -2,4-dichloro or -3,5-dichloro.

[0084] In some embodiments, the compound is selected from the group consisting of:

[0085]

[0086] or a pharmaceutically acceptable salt, hydrate, or solvate thereof. As used herein, the terms “treating'’ or “to treat'’ each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0087] A “subject in need thereof as utilized herein refers to a subject in need of treatment for neurodegenerative diseases and / or disorders, such as Alzheimer’s disease. The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. In some embodiments, the treated subject may be a mammalian subject. Although the methods disclosed herein are particularly intended for the treatment of humans, other mammals are included. By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, mice, rats and pigs.

[0088] As used herein, the term "disorder" refers to a condition in which there is a disturbance of normal functioning. A "disease" is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person. Sometimes the term is used broadly to include injuries, disabilities, syndromes, symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts these may be considered distinguishable categories. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein.

[0089] Neurodegenerative diseases and / or disorders may include, but are not limited to, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, cerebral palsy, stroke / ischemic brain damage, and migraine headaches.

[0090] In some embodiments, the neurodegenerative disease and / or disorder is Alzheimer’s disease.

[0091] In some embodiments, the subject may suffer damage from amyloid-P aggregation and toxicity7. For example, in some embodiments, the subject has oxidative stress damage induced by amyloid-P aggregation.

[0092] In some embodiments, the subject has ferroptosis-induced neurodegeneration. The ferroptosis-induced neurodegeneration may be induced by GPx4 inhibition. In some embodiments, the ferroptosis-induced neurodegeneration may be induced by glutamate / cystine antiporter (x'c) system inhibition.

[0093] Optionally, the disclosed compounds and pharmaceutical compositions may be administered with additional therapeutic agents, optionally in combination, in order to treat diseases and disorders, such as Alzheimer’s disease, to a subject in need thereof. In some embodiments, one or more additional therapeutic agents are administered with the disclosed compounds and pharmaceutical compositions, where the additional therapeutic agent is administered prior to, concurrently with, or after administering the disclosed compounds and pharmaceutical compositions. In some embodiments, the disclosed pharmaceutical compositions are formulated to comprise the disclosed compounds and further to comprise one or more additional therapeutic agents.

[0094] As used herein the term “effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) for treating neurodegenerative diseases and / or disorders, such as Alzheimer’s disease. The compounds may be administered to a subject in an effective amount in such that amyloid-P aggregation is inhibited. In some embodiments, the effective amount of the compound results in reduction or elimination of oxidative damage induced by amyloid-P aggregation. In some embodiments, the effective amount of the compound results in neuroprotective effects against ferroptosis-induced neurodegeneration, such as in neurodegeneration induced by GPx4 inhibition.

[0095] An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

[0096] In some embodiments of the disclosed treatment methods, the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. In some embodiments, the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg. 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. Minimal and / or maximal doses of the compounds may include doses falling within dose ranges having as end-points any of these disclosed doses (e.g., 2.5 mg - 200 mg).

[0097] In some embodiments of the disclosed treatment methods, a minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000. 10000, 15000, or 20000 ng / kg body weight of the subject. In some embodiments, a maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000. 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. Minimal and / or maximal dose levels of the compounds for achieving therapy in the disclosed methods of treatment may include dose levels falling within ranges having as end-points any of these disclosed dose levels (e.g., 500 - 2000 ng / kg body weight of the subject).

[0098] A typical daily dose may contain from about 0.01 mg / kg to about 100 mg / kg (such as from about 0.05 mg / kg to about 50 mg / kg and / or from about 0.1 mg / kg to about 25 mg / kg) of each compound used in the present method of treatment.

[0099] Compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg of each compound individually or in a single unit dosage form, such as from about 5 to about 300 mg, from about 10 to about 100 mg, and / or about 25 mg. The term '‘unit dosage form” refers to a physically discrete unit suitable as unitary dosages for a patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient. Oral administration is an illustrative route of administering the compounds employed in the compositions and methods disclosed herein. Other illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subject and the caregiver.

[0100] In some embodiments, the compound is administered orally.

[0101] In some embodiments, the brain / plasma concentration of the compound in the subject has a ratio of at least 0.3, at least 0.5, at least 0.75, at least 1.3, at least 1.5, at least 1.75, or at least 1.9, from about 0.25 h to about 2 h after oral administration.

[0102] In some embodiments, the compound is administered intravenously.

[0103] In some embodiments, the brain / plasma concentration of the compound in the subject has a ratio of at least 0.3, at least 0.4, or at least 0.5, from about 0.25 h to about 2 h after intravenous administration.

[0104] The compounds and compositions disclosed herein may be administered in methods of treatment as known in the art. Accordingly, various such compounds and compositions can be administered in conjunction with such a method in any suitable way. For example, administration may comprise oral, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof.

[0105] Another aspect of the present disclosure provides a method of inhibiting amyloid-P aggregation and / or reducing amyloid-P aggregation-induced toxicity, the method comprising contacting amyloid P with the compound disclosed herein. In some embodiments, amyloid P is contacted with the compound in vitro or ex vivo. In some embodiments, amyloid P is contacted with the compound in vivo.

[0106] Compounds

[0107] Another aspect of the present disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof:

[0108]

[0109] In some embodiments,

[0110] R1is H or methylene;

[0111] R2is halogen;

[0112] x is 0-4;

[0113] L is - O- (CH2)n-, -CH2-N+HRA-CH2- -(CH2)n-,-N(RA)-, or -N(RA)-(CH2)n-; RAis C1-6alkyl; or RAtogether with R1form -(CH2)m-;

[0114] n is 0-3;

[0115] m is 1-2;

[0116] M is a counterion; and

[0117] y is 0 or 1.

[0118] In some embodiments.

[0119] R1is H or methylene;

[0120] R2is halogen;

[0121] x is 0-2;

[0122] L is — (CH2)n—,-N(RA)-, or -N(RB)-(CH2)n-;

[0123] RAis C1-6alkyl;

[0124] RBtogether with R1form -CH2-;

[0125] n is 0-2

[0126] M is a counterion;

[0127] y is 0 or 1; and

[0128] with the proviso that

[0129] when L is -N(RB)-(CH2)n- and n is 2, R2is not -3,5-dichloro.

[0130] In some further embodiments, L is -(CH2)n- and n is 2 or 0.

[0131] In some further embodiments, L is -NMe- In some further embodiments, R1is H.

[0132] In some further embodiments, L is -N(RA)-CH2-, and RAtogether with R1form -CH2-

[0133] In some further embodiments, R2is -Cl and x is 2.

[0134] In some further embodiments, R2is -2,4-dichloro or -3.5-dichloro.

[0135] In some further embodiments, the compound is selected from the group consisting of:

[0136]

[0137] or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0138] In some embodiments, the compounds disclosed herein may be formulated as pharmaceutical compositions comprising any of the compounds disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0139] The disclosed compounds and compositions comprising the same may exhibit one or more biological activities. In some embodiments, the disclosed compounds inhibit amyloid-0 aggregation. In some embodiments, the disclosed compounds protect neuronal cells from A0 intracellular aggregation and toxicity. In some embodiments, the disclosed compounds may inhibit the A0 aggregate toxicity by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 10 pM, 1 pM, 0.1 pM, 0.05 pM, 0.01 pM. 0.005 pM. 0.001 pM. or less. In some embodiments, the disclosed compounds may inhibit amyloid-0 aggregation by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 10 pM, 1 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.005 pM, 0.001 pM, or less. Concentration ranges also are contemplated herein, for example, a concentration range bounded by end-point concentrations selected from 0.001 pM, 0.005 pM, 0.01 pM, 0.5 pM, 0.1 pM, 1.0 pM, 10 pM, 100 pM, 150 pM, 200 pM, and 250 pM.

[0140] In some embodiments, the disclosed compounds scavenge oxygen-free radical species. Therefore, in some embodiments, the disclosed compounds reduce the effect of reactive species, protect nerve cells from oxidative stress toxicity, and reduce neurodegeneration for AD patients. In some embodiments, the capacity of the compounds to scavenge oxygen-free radical species is evaluated using the oxygen radical absorbance capacity (ORAC). The disclosed compounds may have an ORAC antioxidant unit of at least 0.5, at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5. at least 4.0, or at least 4.5.

[0141] In some embodiments, the disclosed compounds display anti-ferroptotic capacity in a cell. In some embodiments, the anti-ferroptotic capacity of the disclosed compounds is evaluated in the HT22 neuronal cell line in the presence of ferroptosis inducers, such as glutamate, erastin, and RSL3. In some embodiments, the disclosed compounds have neuroprotective effect against ferroptosis-induced neurodegeneration, such as ferroptosis-induced neurodegeneration resulting from GPx4 deficiency.

[0142] In some embodiments, the disclosed compounds permeate through the blood-brain barrier (BBB). In some embodiments, the disclosed compounds exhibit high BBB passive permeability potential and brain / plasma ratios in the in vivo models. In some embodiments, the brain / plasma concentration of the compound in the subject has a ratio of at least 0.3, 0.5, 0.75, 1.3, 1.5, 1.75, or 1.9, from about 0.25 h to about 2 h after oral administration.

[0143] Pharmaceutical Compositions

[0144] Another aspect of the present disclosure provides a pharmaceutical composition comprising the compound disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent. For example, the pharmaceutical composition may comprise a therapeutically effective amount of the compound as disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0145] The compounds employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein. Such compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed compound, which effective amount is related to the daily dose of the compound to be administered. Each dosage unit may contain the daily dose of a given compound, or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound to be contained in each dosage unit can depend, in part, on the identity’ of the particular compound chosen for the therapy and other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures. In some embodiments, the compounds disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg). The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg / kg body weight (preferably about 0.5 to about 500 mg / kg body weight, more preferably about 50 to about 100 mg / kg body weight). In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 nM, 0.005 nM, 0.01 nM, 0.5 nM, 0.1 nM, 1.0 nM, 10 nM, and 100 nM (e.g, 0.1 nM - 1.0 nM).

[0146] It is understood by those skilled in the art that dosage amount will vary with the activity of a particular inhibitor compound, disease state, route of administration, duration of treatment, and like factors well-known in the medical and pharmaceutical arts. In general, a suitable dose will be an amount which is the lowest dose effective to produce a therapeutic or prophylactic effect. If desired, an effective dose of such a compound, pharmaceutically acceptable salt thereof, or related composition may be administered in two or more sub-doses, administered separately over an appropriate period of time.

[0147] In some embodiments, a pharmaceutical composition comprising the compound of as disclosed herein and a pharmaceutically suitable carrier, diluent, or excipient is provided.

[0148] The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 0.5 to 500 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 1 to 100 mg. The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg / kg body weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.5 to about 500 mg / kg body weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 50 to about 100 mg / kg body weight. In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 nM, 0.005 nM. 0.01 nM. 0.5 nM, 0.1 nM, 1.0 nM. 10 nM, and 100 nM (e.g, 0.1 nM - 1.0 nM).

[0149] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0150] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyd alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0151] Suitable diluents may include pharmaceutically acceptable inert fdlers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

[0152] Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, com starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof. Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0153] The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.

[0154] Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

[0155] Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

[0156] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis.

[0157] Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

[0158] For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are in some embodiments applied as a topical ointment or cream. When formulated in an ointment, the compound may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

[0159] Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

[0160] Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

[0161] Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (z. e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

[0162] Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.

[0163] Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0164] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient: and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0165] Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch: or acceptable

[0166] 10 wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.

[0167] Optionally, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with additional therapeutic agents, optionally in combination, in order to treat diseases and disorders. In some embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the disclosed compounds or with pharmaceutical compositions comprising the disclosed compounds, where the additional therapeutic agent is administered prior to, concurrently with, or after administering the disclosed compounds or the pharmaceutical compositions comprising the disclosed compounds. In some embodiments, the disclosed pharmaceutical composition is formulated to comprise the disclosed compounds and further to comprise one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating diseases and disorders.

[0168] Methods of preparing pharmaceutical formulations or compositions include the step of bringing a disclosed compound into association with a carrier and, optionally, one or more additional adjuvants or ingredients. For example, standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

[0169] Regardless of composition or formulation, those skilled in the art will recognize various avenues for medicament administration, together with corresponding factors and parameters to be considered in rendering such a medicament suitable for administration. Miscellaneous

[0170] Unless otherwise specified or indicated by context, the terms "a”, "an", and "the" mean “one or more.’’ For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0171] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0172] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0173] The phrase “such as” should be interpreted as “for example, including.” Moreover the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0174] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.” All language such as “up to,’' “at least,'’ “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0175] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0176] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0177] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0178] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. EXAMPLES

[0179] Compounds 1 and 2 (Figure 1) were previously identified with high potency in cellular models expressing SOD1 with mutations associated with protein aggregation in amyotrophic lateral sclerosis (ALS). These compounds are structurally similar to edaravone, an ALS-approved drug, known for its free-radical scavenging capacity.15In a recent study, edaravone also demonstrated promising therapeutic potential for AD by decreasing Ap aggregation and attenuating A -induced oxidative stress toxicity.16Therefore, with an approach based on small molecules capable of permeating the BBB, targeting Ap aggregation, and minimizing the effects of oxidative stress damage, we aimed to create a synergistic effect that could lead to more efficient therapeutics for AD using these compounds. The present disclosure describes in vitro SAR and in vivo PK / PD studies that allowed us to identify arylpyrazolones as compounds for the development of novel AD therapeutics.

[0180] Results and Discussion

[0181] Chemistry. Compounds 1 and 2 were synthesized following the published protocol.12’13The SAR was expanded through the design of new compounds based on the structures of 1 and 2. The pyrazolone ring, which was previously identified as crucial for the activity of the compounds, w as retained in the new structures. A series of compounds was designed in which modifications were made to the length and lipophilicity of the linker between the pyrazolone and the phenyl ring or incorporated a cyclic rigid ring between the two structures. Additionally, modifications to the substituent position on the phenyl ring were also evaluated. The newly designed compounds (3-7, Figure 1) were obtained following the synthetic route illustrated in Scheme 1.

[0182] A Wittig reaction with the respective commercially available benzaldehydes (8a and 8b) gave the y-5-unsaturated-P-ketoesters 9a and 9b, which were reduced with hydrogen and a Pd / C catalyst and converted to pyrazolones 3 and 4 with hydrazine. Compound 5 was made by the reaction of ethyl 3,5-dichlorobenzoate (10) with ethyl acetate and sodium hydride under reflux, giving 11, which was allowed to react with hydrazine to produce 5. The reaction of N-methylaniline (12) with ethyl 3-chloro-3-oxopropionate furnished intermediate 13, which was allowed to react with hydrazine to yield the tertiary amine pyrazolone derivative 6. Cyclic analogue 7 was obtained starting from commercially available 2,4-dichloroaniline (14), which was allowed to react with ethyl acrylate to give tertiary amine 15. The reaction of 15 with titanium (IV) chloride gave cyclic intermediate 16. Final compound 7 was obtained by the reaction of 16 with hydrazine. Scheme 1. Synthesis of arylpyrazolone derivatives 3-7

[0183]

[0184] Reagents and conditions: (a) ethyl 3-oxo-4-(triphenylphosphoranylidene)butanoate, toluene, reflux, 12 h; (b) Pd / C, H2, EtOH, rt, 6 h; (c) NH2NH2, EtOH, rt, 12 h; d) ethyl acetate, NaH, THF. reflux. 12 h; (e) NH2NH2, EtOH, reflux, 12 h: (1) ethyl 4-chloroacetoacetate, Et3N, EtOAc, 10 °C, 12 h; (g) NH2NH2, EtOAc, rt-reflux, 12 h; (h) ethyl 2-bromoacetate, DIPEA, DMF, 90 °C, 12 h; (i) TiCl4, TEA, DCM, 0 °C, 6 h; (j) NH2NH2, EtOH, rt, 30 min Evaluation of the protective effect of arylpyrazolones against intracellular Ap aggregation. The presence of insoluble Ap peptide plaques in the brains of patients is one of the major hallmarks of AD and is associated with disease onset and progression.18As postulated by the A cascade hypothesis, the cleavage of the transmembrane amyloid precursor protein (APP) by the coordinated activity7of P- and y-secretases leads to the formation of Ap peptides that undergo conformational changes and promote the formation of insoluble toxic plaques involved in the disease pathology.10

[0185] The MC65 human neuroblastoma cell line is designed to express the C99 - C terminal fragment of APP under the control of a tetracycline-sensitive promoter.19In the absence of tetracycline, the C99 fragment is expressed, promoting an increase in intracellular Ap peptide, leading to the formation of aggregates and cell death after 3 days.19Hence, the MC65 cell line is considered a good in vitro model to assess the small-molecule capacity for protecting neuronal cells from A0 intracellular aggregation and toxicity.

[0186] Using MC65 cells as an in vitro model, we screened a small library of aryl pyrazoles (Figure 1) for their protective capacity against A toxicity. The cells were treated with the vehicle or compounds in media containing tetracycline (100% survival), or with the vehicle (0-10% survival) or the compounds in media without tetracycline. The activity’ of edaravone was also screened and compared with our compounds.

[0187] The results of the compound screen against A aggregate toxicity are summarized in Table 1. We started our screen by evaluating the protective capacity of two compounds previously reported by us to have potent in vitro activity against SOD1 aggregate-induced toxicity (1 and 2).12 13The screening results revealed that both compounds exhibited submicromolar ECso values, 0.27 and 0.54 pM for 1 and 2, respectively. Performing an oxygen-to-carbon conversion in the linker between the benzyl ring and the pyrazolone of 1 to increase the lipophilicity (3) led to a 1.7-fold loss in potency. Additionally, changing the chlorine substitution pattern on the benzyl ring to para and ortho (4) further decreased the compound’s activity. Removing the linker between the phenyl ring and the pyrazolone moiety (5) eliminated the compound’s activity in this assay. Reducing the linker size of 2 and removing the phenyl ring chlorine atoms also yielded a non-active compound (6). Insertion of a conformationally restricted linker, in the form of a hexahydro-3 / 7-py razolo|4.3-c|py ridin-3-one biaryl structure (7). also eliminated the activity of the compound.

[0188] The most potent compounds were 7-14 fold more potent than edaravone, an ALS-approved drug with known activity against A aggregation and oxidation toxicity.16In summary, our screen allowed us to identify a series of arylpyrazolone analogues with promising biological activity against induced Ap aggregation toxicity. Furthermore, the initial SAR provided some guidelines to take into consideration for future compound design. Indeed, we observed that changing the phenyl ring substitution pattern or increasing the hydrophobicity of the linker betw een the phenyl ring and pyrazolone moiety is tolerated. We also observed that removing the linker or constricting the linker has a considerable unfavorable impact on the compound’s activity. Table 1. EC50 values against Ap aggregation-induced toxicity in MC65 cells, ORAC antioxidant units, EC50 values obtained in the anti-ferroptotic assay in HT22 cells, and calculated lipophilicity (cLogP) of arylpyrazolone compounds 1-7 and edaravone.

[0189] Glutamate, erastin, and RSL3 were used to induce ferroptosis in HT22 cells.

[0190] ECso A EC50 glutamate EC50 Erastin EC50 RSL3 Compound ORACacLogPb(jiM) (gM) (jiM) (jiM) Edaravone 3.7± 0.15 4.34 ± 0.24 >30 >30 12.3 ± 0.14 1.64

[0191] 1 0.27 ± 0.006 4.15 ± 0.13 16.8 ± 1.8 >30 4.1 ± 0.92 2.59 2 0.54 ± 0.05 3.10 ± 0.26 24 ± 6.0 >30 4.8 ± 0.71 2.62 3 0.44 ± 0.01 3.34 ± 0.28 >30 >30 3.8 ± 0.50 3.15 4 0.56 ± 0.03 3.32 ± 0.03 >30 >30 3.9 ± 1.3 3.15 5 ND 2.09 ± 0.15 >30 >30 8.0 ± 2.4 1.73 6 ND 0.56 ± 0.02 Inactive Inactive Inactive

[0192] 7 ND 3.73 ± 0.30 >30 >30 18.1 ± 1.1 2.02aV alues normalized to the assay control (Trolox);

[0193] hCalculated using SwissADME software (http: / / www.swissadme.ch / index.php) ND - Nondefined

[0194] Arylpyrazolones cell-free profiling of free radical scavenging capacity and evaluation of cellular anti-ferroptotic activity. Due to the presence of transition metals with high redox potential as well as the high levels of unsaturated fatty acids, energy demand and oxygen consumption, nerve cells are very susceptible to oxidative stress damage.20However, an efficient antioxidant biological network normally protects nerve cells from oxidative stress toxicity. During the development of AD, due to aging or other pathological changes, such as Ap plaque accumulation, the antioxidant detoxifying system response is impaired, allowing the accumulation of free radicals that exacerbate neurodegeneration and disease progression.17Hence, compounds with the capacity to minimize the effects of reactive species could reduce neurodegeneration and provide therapeutic benefits to AD patients.

[0195] Edaravone is an ALS-approved drug known for its free radical scavenging capacity. In the last decade, we developed a series of pyrazolone compounds structurally similar to edaravone that demonstrated the capacity to mimic the enol form that is believed to contribute to the radical scavenging capacity of this drug (Figure 2).15

[0196] To evaluate the antioxidant potential of our compounds, we screened their capacity to scavenge oxygen-free radical species using the oxygen radical absorbance capacity (ORAC) assay. This assay measures the fluorescent signal of a probe (fluorescein), which decreases in the presence of reactive oxygen species (ROS).21This assay was performed under the physiologically relevant conditions of pH 7.4 and 37 °C and, used 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) as a ROS generator, which upon thermal decomposition forms peroxyl and alkoxyl free radicals that mimic biologically relevant free radical species. The ability of the compounds to quench the free radical species present in the solution was evaluated by assessing their capacity to delay the loss of the probe fluorescent signal. The antioxidant capacity of the screened compounds was then compared with the antioxidant capacity of the known antioxidant drug edaravone.

[0197] The ORAC results are presented in Table 1. Analyzing the data from our aryl pyrazolone compounds, we observed that decreasing the lipophilicity and size of the linker between the pyrazolone moiety and the benzyl ring corresponded to a significant decrease in the radical scavenging capacity of the compounds, as observed in 5 and, to a higher extent, in 6. However, increasing the lipophilicity of the linker between the benzyl ring and pyrazolone moiety increased the antioxidant capacity of the compounds, as observed in compounds 2, 3, and 4 and to a greater extent in 1, whose radical scavenging capacity was not significantly different from that of edaravone. Conformational restriction of the linker to form a hexahydro-3H-pyrazolo[4,3-c]pyridin-3-one biaryl structure (7) was tolerated and had the second-best radical scavenging capacity7in this series. Overall, our data suggest that our compounds present a radical scavenging capacity that may allow them to protect biological systems from oxidative stress. Furthermore, the most promising compounds also present a radical scavenging capacity comparable to that of the antioxidant drug edaravone.

[0198] To evaluate the antioxidant potential of our aryl pyrazolone compounds in a biological system, we evaluated their anti-ferroptotic capacity7in a cellular model. Ferroptosis is a recently described cell death process characterized by the iron-dependent accumulation of phospholipid peroxides that contribute to the loss of cell membrane integrity.22Evidence also demonstrates that mitochondrial dysfunction, the loss of activity^ of biological systems involved in lipid repair and antioxidant detoxification, such as the cystine / glutamate antiporter system (x’c) involved in glutathione synthesis, and glutathione peroxidase 4 (GPx4), contribute to ferroptosis induced cell death.22Recently, research suggests that ferroptosis is one of the biological mechanisms contributing to neurodegeneration in AD and that this process can be reduced by the use of antioxidants such as edaravone.23’24Hence, these observations suggest that the use of compounds with antioxidant activity7could have a beneficial effect by decreasing ferroptosis-induced neurodegeneration in AD. The anti-ferroptotic potential of our compounds was evaluated using the HT22 neuronal cell line, a well-described model to study ferroptosis-induced neurotoxicity.25-26The neuroprotective activity of the compounds was evaluated in the presence of ferroptosis inducers glutamate, erastin (inhibitors of the glutamate / cy stine antiporter (x’c) system), and RSL3 (GPx4 inhibitor).27The results with the compounds were compared to the activity of the known antioxidant edaravone and are shown in Table 1.

[0199] Compound 6, the compound exhibiting the lowest radical scavenging capacity in the ORAC assay was inactive in the cellular assay. We observed that most of the tested compounds and edaravone demonstrated mild anti-ferroptotic potential in cells treated with RSL3 (GPx4 inhibitor). However, in the presence of erastin or glutamate, our compounds exhibited low protective potential. Compounds 1-5 had higher protective capacity in the presence of RSL3 than edaravone. These results suggest that the higher hydrophobicity of these compounds may induce a greater cell membrane passive permeability thereby increasing their intracellular concentration, leading to a higher cellular activity despite lower or comparable radical scavenging activity when compared to edaravone in the ORAC assay. Indeed, in line with these observations, when comparing compounds 3 and 4, in which the linker between the phenyl ring and the pyrazolone moiety differs from 1 by substitution of the oxygen atom with a methylene group to increase lipophilicity7, we observed comparable cellular activity7despite their lower activity in the ORAC assay. Conformational restriction of the linker in 7 led to a considerable loss in anti-ferroptotic potential, despite exhibiting the third-best radical scavenging capacity in the ORAC assay and lipophilicity higher than 5, which also exhibited considerably lower radical scavenging activity7but more than two-fold higher cellular activity'.

[0200] In summary7, we report a series of arylpyrazolones with neuroprotective effect against ferroptosis-induced neurodegeneration, most specifically in toxicity induced by GPx4 inhibition. Additionally, our results suggest that the most active arylpyrazolones are potent inhibitors of lipid peroxidation, a process also triggered by RSL3-dependent GPx4 inhibition.28In recent reports, researchers observed that GPx4-deficient mice exhibited severe neurological deficits and cognitive impairment.29In another report, mice treated with Ap also exhibited decreased levels of GPx4 in their brains.30Furthermore, ferroptosis was shown to play a key role in MC65 neuronal cell death induced by intracellular Ap aggregation.19These results suggest that arylpyrazolone compounds, such as 1-4, could also protect neurons in AD from ferroptosis-induced neurodegeneration resulting from GPx4 deficiency and, based on the protective results of these compounds in MC65 cells, also against ferroptotic death promoted by Ap aggregation-induced toxicity. These compounds also demonstrated a 3-fold higher protective capacity in these assays than edaravone, a drug with the capacity’ to protect cellular models from A -induced death through the ferroptosis axis.23Compounds 1-4 also exhibited promising neuroprotective potential against Ap intracellular aggregation toxicity’ and protective antioxidant capacity7. The activity of the most promising candidates against Ap intracellular toxicity can be partially explained by the capacity of the compounds to minimize the oxidative damage induced by aggregate formation. However, in line with other studies, we observed considerable differences among the activities of 1-4 in the anti-ferroptosis activity assay (micromolar range) and the activity in the Ap intracellular toxicity7(nanomolar range) assay.31Furthermore, in the Ap intracellular toxicity assay, no activity was observed for compounds 5 and 7, despite their promising radical scavenging capacity and slightly better or comparable anti-ferroptotic activity than edaravone. Therefore, this observation suggests that the neuroprotective activity of our arylpyrazolones also could be, in part, justified by their capacity7to modulate additional biological pathways and / or engage protein targets with relevant activity against Ap aggregate-induced toxicity.

[0201] In vitro and in vivo evaluation of blood-brain barrier (BBB) permeability and pharmacokinetic profile of the most promising arylpyrazolone derivatives. The success of drugs developed for diseases affecting the central nervous system (CNS), such as AD or other neurodegenerative diseases, is dependent on their capacity to permeate through the BBB.'3The BBB is a highly specialized barrier formed by specific tight junctions between brain endothelial cells that restricts access to the brain of toxic, as well as therapeutic, agents.33Hence, drug candidates for CNS-related diseases need to be able to permeate this barrier and accumulate in the brain in sufficient quantities to engage their targets and promote a therapeutic effect. Passive diffusion through physiological membranes, including the BBB, is the most common and less energetically demanding process of drug permeation.33Therefore, the prediction of BBB passive permeability using biomimetic methods is made early in the drug discovery process to fine-tune the properties of candidates and ensure the selection of the most promising drug candidates for CNS-related diseases. One of the most common methods to evaluate smallmolecule BBB permeability is the parallel artificial membrane permeability assay (PAMPA-BBB), in which the passive diffusion of small-molecules through a membrane mimicking the physicochemical environment of the BBB is evaluated.34Following this rationale, we performed PAMPA-BBB assays to predict the passive permeability of the most potent arylpyrazolones (1-3), and the results were compared with edaravone, known for its capacity to permeate the BBB (Table 2).

[0202] Our data revealed that arylpyrazolones 1-3 exhibited BBB passive permeability higher than that of edaravone and in the range of verapamil, a compound with high BBB passive permeability that we used as a control in this assay. Furthermore, lipophilicity was a parameter believed to contribute to the observed permeability. The increase in lipophilicity of 1 and 3 paralleled an increase in passive permeability. However, there was a slight decrease in passive permeability for 2, despite its higher hydrophobicity. Following these observations, we calculated the physiochemical properties of 1-3 and edaravone including topological polar surface area (TPSA), molecular weight (MW), number of hydrogen bond donors (NHBD), and number of rotatable bonds (NRB). which are parameters known to closely impact the passive permeability of small molecules through the BBB.35’36Lipophilicity compensated for the increase in TPSA for 1-3, which exhibited higher permeability than edaravone; however, edaravone presented a more favorable BBB permeability profile in terms of TPSA.35'36The larger NHBD and NRB of 2 likely negatively impacted its passive permeability relative to 1 despite the higher lipophilicity and lower TPSA of 2.

[0203] Table 2. PAMPA-BBB passive permeability (Pe) for edaravone, arylpyrazolone derivatives, and for the assay controls verapamil (high permeability) and theophylline (low permeability). Lipophilicity (Log P), topological polar surface area (TPSA), molecular weight (MW), number of hydrogen bond donor groups (NHBD), and number of rotatable bonds (NRB) of the evaluated compounds

[0204] Pe TPSA MW

[0205] Compound Log PeaLog Pb, NHBDbNRBb(106cm / s) (A2)b(g / mol)bEdaravone 17.43 ± 3.20 -4.76 1.64 32.67 174.20 0 1

[0206] 1 23.46 ± 2.15 -4.63 2.59 57.88 259.09 2 3 2 20.80 ± 0.37 -4.68 2.62 53.09 287.16 3 4 3 25.68 ± 1.96 -4.59 3.15 48.65 257.11 2 3 Verapamil 25.41 ± 1.87 -4.60....

[0207] Theophylline 0.22 ± 0.02 -6.66....

[0208] aPrediction of BBB passive permeability scale. No permeability LogPe < -6.14; low permeability -6.14< LogPe <-5.66: medium permeability -5.66< LogPe<-5.33; high permeability LogPe > -5.33.

[0209] bValues were calculated using the Swiss ADME website

[0210] (http: / / www. s wissadme. ch / index. php) Despite the use of PAMPA to predict BBB small molecule permeation, the major limitation of this technique is that active transport and efflux by P-gly coprotein (P-gp) are not assessed in this assay.37Furthermore, in in vivo systems, this parameter can also be influenced by the pharmacokinetic-pharmacodynamic (PKPD) profile, which plays a key role in drug bioavailability7and significantly impacts their potency and efficacy.18Hence, the determination of in vivo PKPD profile and BBB permeation is crucial for validating drug activity7and specific tissue engagement. Based on the above results, we selected 1, the candidate presenting the highest in vitro potency and good BBB passive permeability7and evaluated it for in vivo pharmacokinetics and BBB permeability. In these studies, mice were administered a single dose of 1 intravenously (iv, 5 mg / kg) or orally (po, 30 mg / kg), and the amount of 1 in the plasma and brain tissues of the animals was quantified by ESI mass spectrometry at specific timepoints (Figure 3 and Tables 4 and 5 and Figure 7). From the experimental data, the pharmacokinetic parameters of 1 were determined (Table 3).

[0211] Table 3. Mouse plasma pharmacokinetic profile of 1

[0212] AUCiast CL VssCo / Cmax

[0213] ti / 2(h) traax(h) F (%) (hr*ng / mL) (mL / min / kg) (L / kg) (ng / mL) iv (5 mg / mL) 294.55 1.15 275.06 9.98 1277.49 po (30mg / mL) 424.41 2.74 - - 256.40 0.25 24

[0214] Analysis of the data for the animals administered 1 intravenously revealed that the compound was detected in plasma at appreciable levels over the first 2 h and in the brain over the initial hour after compound administration. We also observed a high plasma clearance rate (275.06 mL / min / kg) for iv-administered 1, which was paralleled by a half-life of 1.15 h. These results were in line with previous in vitro results of 1 that revealed a high mouse microsomal clearance and suggested high in vivo hepatic metabolism and low plasma half-life.12’39Oral administration of 1 exhibited a longer half-life (2.74 h) and was detected in plasma and brain tissues for a longer time. Despite appearing quite promising, these results should be interpreted with some caution since the difference between the tw o analyses could be explained by the fact that the dose of 1 given to the animals w as 6 times higher in the oral administration studies as compared with the iv administration studies. Nevertheless, the brain / plasma ratio observed for 1 was considerably higher than the values observed for most marketed CNS drugs (brain / plasma > 0.3), and the observed oral bioavailability (F) of 24% is within the range of most drugs during optimization programs.40'42Furthermore, our in vitro studies also suggested lower human microsomal clearance predicting low er hepatic clearance in humans as compared with mice, which could increase the drug plasma half-life and therefore yield higher bioavailability. Indeed, these differences between species are not unusual and reflect interspecies differences in liver metabolism.43

[0215] The overall results favor the selection of arylpyrazolone, such as 1, AD treatment. These compounds present not only promising in vitro activity7against pathologies associated with AD progression but also an excellent starting pharmacological profile. Indeed, the compound exhibited a high BBB passive permeability potential and good brain / plasma ratio in our in vivo studies despite the lower half-life of the compound in plasma.

[0216] Conclusion. In this work, we report a series of arylpyrazolone derivatives with promising activity against Ap aggregate toxicity and oxidative stress insults, revealing a multimodal mechanism of action, which increases their therapeutic potential. The most active compounds also exhibited excellent BBB passive permeability' and a strong capacity for permeating the BBB in vivo, presenting a B / P ratio > 1 over more than 2 h and a promising bioavailability (24%) after oral administration. These characteristics would ensure the accumulation of the compound in brain tissues at a concentration that could allow for a therapeutic effect, despite a relatively fast clearance and low plasma half-life. In summary, we demonstrated the potential of our ary lpyrazolone compounds for the development of novel and potentially more effective therapies for AD either on their own or in combination with other treatments.

[0217] Experimental

[0218] Chemical Synthesis

[0219] General experimental conditions. The solvents used in the reactions were from analytical grade and obtained from Millipore Sigma and Fisher Scientific. The commercially available reagents and starting materials were purchased from TCI, Millipore Sigma, and Alfa Aeser and used without further purification unless stated otherwise. The reactions were performed using magnetic stirring plates and monitored by thin layer chromatography (TLC) on precoated silica gel plates (silica gel 60 F254). with visualization by UV light (254 nm). The crude compounds were purified by flash column chromatography using prepacked silica gel cartridges (230-400 mesh, 40-63 mm; RediSep Silver- Teledyne ISCO) and a Teledyne ISCO Combiflash RF+ or Combiflash Nextgen 300+ using the solvent mixtures stated for each synthesis as mobile phase.

[0220] The 'H and13C nuclear magnetic resonance (NMR) spectra were acquired at room temperature on a Bruker Avance III 500 spectrometer (*H at 500 MHz and13C at 126 MHz) equipped with a DCH CryoProbe or on a Bruker Avance III HD system ('H at 500 MHz and13C at 126 MHz) equipped with a BBO Prodigy probe. The chemical shifts are expressed in 8 (ppm) values using residual deuterated solvents as the internal reference. The signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), pentate (p), multiplet (m). The coupling constants (J) are quoted to the nearest 0.1 Hz. The purity of the final compounds was determined by liquid chromatography-mass spectrometry’ (LC-MS) on a Waters Acquity H Plus UPLC connected to a diode array and a Waters Xevo TQ-S micro triple quadrupole mass spectrometer using a flow rate of 0.4 mL / min and a gradient elution of acetonitrile (0.1% formic acid) in water (0.1% formic acid) over 7 min as follows: 10% acetonitrile over 30 s, acetonitrile gradient from 10-100 % from 30 s to 5 min, acetonitrile 100% from 5-6 min, acetonitrile gradient from 100-10% from 6-6.2 min and then 10% acetonitrile was maintained until the end of the program. The purity of all final compounds was >95%.

[0221] The high-resolution mass spectrometry (HRMS) data of the newly synthesized final compounds was obtained using an Agilent 1100 MSD instrument with methanol as solvent and acquired in the positive or negative mode as specified in the protocol.

[0222] The HPLC chromatograms of compounds 3-7 are shown in Figures 8-12.

[0223] General procedure A for Wittig reaction of benzaldehydes and ethyl 3-oxo-4-(triphenylphosphoranylidene)butanoate. The benzaldehyde (1.14 mmol) and ethyl 3-oxo-4-(triphenylphosphoranylidene)butanoate (1.14 mmol) were added to a round-bottomed flask containing toluene (10 mL). The resulting reaction mixture was heated to reflux under a nitrogen atmosphere overnight. When the reaction was complete, the solvent was removed under reduced pressure. The resulting residue was purified by silica gel column chromatography with hexane and EtOAc (10: 1) as eluent to obtain products 9a and 9b.

[0224] (E )-Ethyl 5-(3,5-dichlorophenyl)-3-oxopent-4-enoate (9a). The title compound was obtained as an off-white solid (0.2 g, 85% yield). 'H NMR (500 MHz, CDCh) 5 11.83 (d, J = 5.0 Hz, 1H), 7.20 (s, 1H), 7.18 (d, J = 15.0 Hz, 1H), 6.32 (d, J= 15.0 Hz, 1H ), 5.09 (s, 1H), 4.15 (q, J = 5.0 Hz, 2H), 1.23 (t, J = 7.5 Hz, 3H);13C NMR (126 MHz. CDCh) 5 172.58, 167.90. 138.41, 135.43, 133.65, 128.82. 125,68. 93.34, 60.47, 14.27.

[0225] (£)-Ethyl 5-(2,4-dichlorophenyl)-3-oxopent-4-enoate (9b). The title compound was obtained as an off-white solid (0.52 g, 63% yield). 'H NMR (500 MHz, CDCh) 5 11.80 (s, 1H), 7.57 (d, J= 15.8 Hz, 1H), 7.35 (d, J= 8.5 Hz, 1H), 7.07 (dd, J= 8.5, 2.1 Hz, 1H), 5.03 (s, 1H), 4.09 (q, J= 7.1 Hz, 2H), 1.16 (t, J= 7.1 Hz, 3H);13C NMR (126 MHz, CDCh) 5 172.60, 168.31. 135.24, 135.04, 132.18, 131.30, 129.87. 127.77, 127.40, 124.77, 60.41, 14.27.

[0226] General procedure B for the hydrogenation of y-8-unsaturated-P-ketoesters. y-5-Unsaturated-P-ketoesters (3.5 mmol) were added to a round-bottomed flask and dissolved in EtOH (8 mL) under argon. After degassing for 15 min, Pd / C (10 mole %) was added to the flask. The reaction was flushed with argon once, evacuated, then filled with hydrogen, and evacuated twice before being backfilled again with hydrogen. The mixture was stirred at room temperature for 2 h. After completion of the reaction, the mixture was filtered through Celite with EtOH and concentrated in vacuo. The crude compound was taken to the next step without further purification.

[0227] General procedure C for the synthesis of pyrazolones from P-ketoesters. To a solution of -ketoesters (0.35 mmol) in EtOH was added anhydrous hydrazine (1.4 mmol). The resulting solution was stirred for 1 h at room temperature. After evaporation of the volatiles, the residue was purified by cry stallization with EtOH to obtain products 3 and 4.

[0228] 5-(3,5-Dichlorophenethyl)-lH-pyrazol-3(2H)-one (3). The title product was obtained as a white solid (62 mg, 70% yield). 'H NMR (500 MHz, (CD3)2CO) 5 10.26 (s, 1H), 7.17 (t, J= 1.9 Hz, 1H), 7.14 (d, J= 2.0 Hz, 2H), 5.26 (s, 1H), 2.86 (ddd, J= 8.5, 6.7, 1.9 Hz, 3H), 2.79 (ddd, J = 8.7, 6.9, 1.9 Hz, 2H);13C NMR (126 MHz, (CD3)2CO) 5 162.1, 145.5, 143.6, 134.4, 127.2, 125.9, 88.6, 34.3, 27.2. HRMS (m / z): [M + H]+calcd for C11H11CI2N2O, 257.0250; found 257.0253.

[0229] 5-(2,4-Dichlorophenethyl)-lH-pyrazol-3(2H)-one (4). The title product was obtained as a white solid (145 mg, 95% yield). 'H NMR (500 MHz, (CD3)2SO) 5 7.57 (d, J= 2.0 Hz, 1H), 7.34 (q, J= 8.2 Hz, 2H), 5.25 (s, 1H), 3.00 - 2.88 (m, 2H), 2.73 (t, J= 7.8 Hz, 2H);13C NMR (126 MHz, (CD3)2SO) 5 137.92, 134.28, 132.39, 132.01. 129.07, 127.79, 32.21, 26.02. HRMS (m / z): [M + H]+calcd for C11H11CI2N2O, 257.0250; found 257.0253.

[0230] Ethyl (3,5-dichlorophenyl)acetate (11). Ethyl 3,5-dichlorobenzoate (10, 1 g, 4.6 mmol) and sodium hydride (0.22 g, 5.5 mmol) were dissolved in THF (5 mL) under nitrogen. To the resulting suspension was added EtOAc (1.56 mL) slowly at room temperature and refluxed overnight. Ammonium chloride solution was added to quench the reaction, then the solvent was removed under reduced pressure. The mixture was extracted with EtOAc, and the organic layer was washed with water and brine. The resulting organic layer was dried over Na2SO4 and the solvent evaporated under reduced pressure to obtain a crude residue that was purified by silica gel chromatography with hexane and EtOAc (20: 1). The keto-enol product (11) was a white solid (110 mg, 92% yield). NMR (500 MHz, CDCh) 5 12.34 (s, 1H), 7.62 (s, 2H), 7.45 (s, 2H), 7.39 (s, 1H), 7.24 (s, 1H), 5.46 (s, 1H), 4.11 (q, J = 15.0, 5.0 Hz. 2H), 4.06 (q, J= 15.0, 5.0 Hz, 2H), 3.79 (s, 2H), 1.18 (t, J = 7.5 Hz, 2H), 1.11 (t, J = 7.5 Hz, 2H);13C NMR (125 MHz, CDCh) 5 190.1, 172.7, 168.1, 166.7, 138.4, 136.4, 135.9, 135.4, 133.3, 130.8, 127.0, 124.5, 89.1, 61.8, 60.8, 45.9, 14.3, 14.1.

[0231] 5-(3,5-Dichlorophenyl)-lH-pyrazol-3(2H)-one (5). To a solution of ethyl 3-(3,5-dichlorophenyl)-3-oxopropanoate (11, 200 mg. 0.77 mmol) in EtOH was added anhydrous hydrazine (0.074 mL, 2.31 mmol). The resulting solution was stirred at reflux overnight. After evaporation of the volatiles, the reaction residue was purified by crystallization with methanol. The product (5) was obtained as a white solid (0.12 g, 72% yield). ’H NMR (500 MHz, CD3OD) 57.62 (t, J= 1.4 Hz. 2H), 7.35 (q. J= 1.7 Hz. 1H).13C NMR (126 MHz. CD3OD) 5 161.00, 144.85, 135.41, 135.09, 126.86, 124.23, 123.26. HRMS (m / z): [M + H]+calcd for C12H7CI2N2O, 228.9937; found 228.9939.

[0232] Ethyl 3-(methyl(phenyl)amino)-3-oxopropanoate (13). In a 50 mL 3-neck flask was added a solution of JV-methylaniline (12, 1.0 g, 9.33 mmol) in EtOAc (15 mL) and Et3N (1.25 mL, 11.20 mmol). The mixture was cooled to 10 °C, and the neat ethyl 3-chloro-3-oxopropionate (1.23 mL, 10.26 mmol) was added over 5 min, maintaining the internal temperature below 15 °C. The mixture was stirred for 1.5 h. After completion of the reaction, the mixture was poured into ice-cold water and stirred for 30 min and then layers separated. The organic layer was washed with aqueous NaHCCL and then dried over Na2SO4, filtered, and the solvent evaporated under reduced pressure. The crude residue was purified by silica gel column chromatography with a mixture of hexane and EtOAc (10:1) to afford ethyl 3-(methyl(phenyl)amino)-3-oxopropanoate (13) as a brown oil (1.8 g, 89%). 'H NMR (500 MHz, CDCh) 57.24 (t. J= 7.7 Hz. 2H), 7.16 (t, J= 7.4 Hz, 1H), 7.08 (dd, J= 7.5, 1.8 Hz, 2H), 3.87 (q, J= 7.1 Hz, 2H), 3.07 (s, 3H), 3.00 (s, 2H), 0.98 (t, J= 7.2 Hz, 3H).13C NMR (126 MHz, CDCh) 5 167.3, 165.5, 143.2, 129.7, 128.0, 127.0, 60.7, 41.2, 37.0, 13.8.

[0233] 5-(Methyl(phenyl)amino)-lH-pyrazol-3(2H)-one (6). To a solution of ethyl 3-(methyl(phenyl)amino)-3-oxopropanoate (13, 300 mg, 1.36 mmol) in EtOH was added anhydrous hydrazine (130 pL. 4.07 mmol). The resulting solution was stirred for 1 h at room temperature. After evaporation of the volatiles, the reaction residue was purified by crystallization with EtOH. The final product was obtained as a brown gummy liquid (6, 212 mg, 83% yield). ’H NMR (500 MHz, CD3OD) 57.35 (dd, J= 8.3, 6.9 Hz, 2H), 7.30 - 7.25 (m, 1H), 7.24 - 7.20 (m. 2H), 3.13 (s. 3H), 2.97 (s. 2H).13C NMR (126 MHz, CD3OD) 5 167.8, 167.6, 143.3, 129.8, 128.2, 127.2, 48.3, 48.1, 48.0, 47.8, 47.6, 47.4, 47.3, 40.0, 36.6. HRMS (m / z): [M +H2O + H]+calcd for C10H14N3O, 208.1088; found 208.1089.

[0234] Diethyl 3,3'-((2,4-dichlorophenyl)azanediyl)dipropanoate (15). 2,4-Dichloroaniline (14, 500 mg, 3.0 mmol), ethyl acrylate (excess amount, 2 mL), and TfOH (15 pL, 10 mol %) were mixed in a round bottom flask, and the reaction mixture was heated under reflux for 24 h. After completion, the solvent was concentrated under reduced pressure. Water was added, and the mixture was neutralized with NaHCCh. The mixture was then extracted with EtOAc, and the organic layer was dried over NazSCk The organic solvent was evaporated under reduced pressure, and the resulting crude residue was purified by silica gel column chromatography with hexane and EtOAc (10:1) to give 15 as a colorless liquid (860 mg, 78% yield). 'H NMR (500 MHz, CDCh) 87.56 (d, J= 5.0 Hz, 1H), 7.36 (dd, J= 10.0. 5.0 Hz, 1H), 7.23 (d, J = 10.0, Hz, 1H), 4.26 (q, J = 7.5 Hz, 4H), 3.59 (t, J = 5.0 Hz, 4H), 2.62 (t, J = 7.5 Hz, 4H), 1.40 (t, J= 7.5 Hz, 6H);13C NMR (126 MHz, CDCls) 5 172.0, 145.1, 132.4, 130.6, 129.6, 127.4, 125.5, 60.6, 48.9, 32.9, 24.3.

[0235] Ethyl l-(2,4-dichlorophenyl)-4-hydroxy-l,2,5,6-tetrahydropyridine-3-carboxylate (16). A solution of diethyl 3,3'-((2,4-dichlorophenyl)azanediyl)dipropanoate (15, 100 mg, 0.277 mmol) in dry DCM was added to TiCL (IM solution in DCM) (0.277 mL, 0.277 mmol) at -5 °C. The mixture was stirred for 2 h at the same temperature. Then tri ethylamine (0.609 mmol, 0.085 mL) was added, and the mixture was stirred for 4 h while keeping the temperature constant. The mixture was treated with saturated NH4CI solution, and then extracted with EtOAc. The combined organic layers were washed with water, dried over Na2SO4, filtered, and the solvent removed under reduced pressure. The crude residue was purified by silica gel column chromatography with hexane and EtOAc (20:1). The product (16) was obtained as a white solid (72 mg, 83% yield). 'H NMR (500 MHz. CDCL) 5 12.1 (s, 1H), 7.38 (d, J= 5.0 Hz, 1H), 7.20 (dd, J= 5.0 Hz, 1H), 7.04 (d, J= 10.0 Hz, 1H), 4.25 (q, J= 5.0 Hz, 2H), 3.73 (t, J= 5.0 Hz, 2H), 3.23 (t, J= 5.0 Hz, 2H), 2.56-2.53 (m, 2H), 1.30 (t, J= 7.5 Hz, 3H);13C NMR (126 MHz, CDCI3) 6 170.9, 170.6, 147.4, 130.5, 129.6, 128.6, 127.7, 121.6, 97.0, 60.7, 48.5, 47.3, 29.7, 14.5.

[0236] 5-(2,4-Dichlorophenyl)-4,5,6,7-tetrahydro-lH-pyrazolo[4,3-c]pyridin-3(2H)-one (7). To a solution of ethyl l-(2,4-dichlorophenyl)-4-hydroxy-l,2,5,6-tetrahydropyridine-3-carboxylate (16, 500 mg, 1.58 mmol) in EtOH / H2O was added anhydrous hydrazine (0.152 mL, 4.75 mmol). The resulting solution was heated to reflux and stirred overnight. After evaporation of the volatiles, the reaction residue was purified by crystallization with MeOH. The final product (7) was obtained as a white solid (0.38 g, 85% yield).JH NMR (500 MHz, CD3OD) 5 7.45 (d, J = 2.4 Hz, 1H). 7.30 (dd, J= 8.7, 2.4 Hz, 1H), 7.23 (d, J = 8.7 Hz. 1H), 3.91 (d, J = 1.3 Hz, 2H), 3.37 (s, 2H), 2.77 (t, J = 5.5 Hz, 2H).13C NMR (126 MHz, CD3OD) 5 147.9, 129.7, 129.1, 127.9, 127.4, 121.8, 48.6, 45.0. HRMS (m / z): [M - H]’ calculated for C12H10CI2N3O, 282.0199; found 282.0200.

[0237] Evaluation of pyrazolone compound protection against Ap intracellular toxicity in MC65 cells. The protective effect against Ap intracellular toxicity was determined as previously described.58Briefly, the MC65 human neuroblastoma cells were maintained in culture in DMEM high glucose supplemented with 10% FBS, 1% antibiotics (including penicillin and streptomycin) and tetracycline (2 pg / mL) and incubated in a humidified atmosphere containing 10% CO2. To perform the assay, the cells were plated at a density of 1 x 105cells per well in 24 well tissue culture plates and then incubated overnight. The medium was removed, and the cells were washed with PBS containing calcium and magnesium (3x), then Opti-minimal essential media (Opti-MEM, Invitrogen) in the presence or absence of tetracycline with the different concentrations of the compounds or vehicle (control) was added. After 3 days, the cell viability was determined using the MTT assay. Optical absorbance was recorded at 570 nm on a SpectraMax M5 microplate reader. The data were obtained from at least four independent replicates, and the results are presented as an average of the ECsos (halfmaximum effective concentration) ± their standard deviation. Results were also verified by visual inspection under the microscope.

[0238] Dose-dependent curves of aryl pyrazolones 1-4 and edaravone on A intracellular aggregation toxicity studies on MC65 cells are shown in Figure 4,

[0239] Oxygen radical absorbance capacity (ORAC) assay. Phosphate buffer (75 mM, pH 7.4) used to dilute a stock solution of fluorescein (5 mM) prepared in ethanol to a working solution of 50 nM, which was covered with aluminium foil before starting the assay. The buffer solution was used to prepare a stock solution of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) (180 mM) that was also covered in aluminium foil. Stock solutions of Trolox (control) and of the tested compounds (5 mM) were prepared in DMSO, and then the initial stock solutions were serially diluted with phosphate buffer to seven working solutions, Trolox (20-80 pM) and tested compounds (1-20 pM). An initial fluorescence reading (excitation 485 nm and emission 528 nm) w as performed after adding 160 pL of the working fluorescein solution (50 nM) and 20 pL of phosphate buffer solution (blank) or 20 pL of the working solutions of Trolox or the working solutions of the tested compounds to a 96-well black plate. Then AAPH stock solution (20 pL,180 mM) was quickly added to each well, and the plate was placed in the plate reader. The fluorescence readings were recorded at each minute over 120 min. The final concentration in each well was 40 nM fluorescein, 18 mM AAPH, serial dilutions [2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 pM] for Trolox and [0.1, 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0 pM] for the tested compounds. The data were acquired on a BioTek Synergy H1MD equipped with a monochromator; the experiments were performed at 37 °C. The fluorescence decay curves were normalized by the maximum value of fluorescence (t = 0 min, blank well), and the areas under the curves (AUC) were determined according to Equation 1, where

[0240] (Equation 1)

[0241]

[0242] fo = initial fluorescence reading at 0 min from the untreated wells (blank) and ft = fluorescence reading at time i. The net AUC is obtained by subtracting the AUC of the blank wells from that of the wells of the compounds at different concentrations. The ORAC unit values of the different test compounds are presented relative to the Trolox ORAC unit values and quantified using Equation 2.

[0243] UCfest compound- UCbiank [Trolox]

[0244] ORAC value = (Equation 2)

[0245] AUClrolox-AUCblank[Test compound]

[0246] AUCbiank = area under the curve of blank; AUCtest compound = area under the curve of the test compound; AUCiroiox = area under the curve of Trolox; [Trolox] = Trolox concentration; and [Test compound] = test compound concentration.

[0247] The individual experiments were performed in triplicate, and the relative ORAC values given for each of the test compounds are presented as an average of three individual experiments ± their standard deviation.

[0248] Evaluation of aryl pyrazolone compound protection against ferroptosis-induced cell death in HT22 cells The anti-ferroptotic potential of the compounds was performed as previously described.75The HT22 mouse hippocampal cells were maintained in DMEM high glucose supplemented with 10% FBS and 1% antibiotics (penicillin and streptomycin) and incubated in a humidified atmosphere containing 10% CO2. To perform the assay, HT22 cells were plated at a density of 5 x 103cells / well in 96-well tissue culture plates and incubated for 24 h. The cells were treated with different concentrations of the compounds alone or in the presence of 5 mM glutamate, 500 nM erastin, or 50-100 nM RSL3 to induce cell death. After 18 h incubation, cell viability was assessed using the MTT assay. Optical absorbance was recorded at 570 nm on a SpectraMax M5 microplate reader. The data were obtained from at least two independent experiments with all samples run in triplicate, and the results are presented as an average of the compounds ECsos ± their standard deviation. Results were also verified by visual inspection under the microscope.

[0249] Dose-dependent curves of aryl pyrazolones 1-4, 5, 7 in anti-ferroptotic toxicity studies on HT22 cells treated with RSL3 and glutamate are shown in Figures 5 and 6.

[0250] Parallel artificial membrane permeability assay-blood-brain barrier (PAMPA-BBB). A solution of 1 xPBS (pH 7.5) was prepared by the dilution of a commercially available 10 xPBS solution. The 10 mM stock solutions of the tested compounds and the controls verapamil (high permeability) and theophylline (low permeability) were prepared in DMSO and then diluted in 1 xPBS to obtain the final solution of the compounds at 250 pM (2% DMSO v / v). A solution of porcine brain lipid (PBL, 20 mg / mL) in dodecane was prepared, and 4 pL of this solution was used to coat the filters on the bottom of the PAMPA donor plates. After air drying for 20 min, the donor plate wells were charged with 250 pL of the solutions of the tested compounds or of the controls. The donor plate was then placed over the acceptor plate in which wells were previously filled with 1 xPBS (2% DMSO v / v) solution. The PAMPA sandwich was closed with parafilm and incubated at 25 °C over 18 h in a humidity-saturated atmosphere with orbital agitation. After incubation, 150 pL of the donor or acceptor well of each compound was collected and transferred to a new plate with low UV absorption at 240 nm (UV star plates). The UV spectra (250-400 nm) of each well were acquired on a BioTek Synergy H1MD equipped with a monochromator at room temperature. The passive permeability (Pe) of the compounds and the controls was determined using Equation 3, where

[0251] (Equation 3) ’

[0252]

[0253] A is the area of the filter of the donor wells (0.21 cm2); t is the incubation time (64800 s); TSS is the time to reach the steady state (usually neglectable because it is very short compared to the total incubation time); VA and VD are the volumes of the acceptor and donor wells, respectively (0.25 cm3); R is the membrane retention factor and is calculated using Equation 4, where

[0254]

[0255] CA (t) is the concentration of the compounds at the final time of incubation on the acceptor well; CD (0) and CD (t) are the concentration of the compounds at the initial time of incubation and at the final time of incubation on the donor well. The permeability of the compounds was considered valid when the recovery rate of the compounds was >75%, and the Pe results are presented as an average of at least three independent experiments, each performed in triplicate.

[0256] Evaluation of the pharmacokinetic and plasma-to-brain distribution profile of 1 in male C57BL / 6 mice. Twenty-seven healthy male C57BL / 6 mice (8-12 weeks old) weighing 22-30 g were placed in groups of three per cage and maintained at a temperature of 22 ± 3 °C and humidity 30-70%, through a 12-h light-dark cycle. The animals were then divided into two groups. Group 1 (12 animals) was administered a formulation of 1 (5 mg / kg) intravenously and Group 2 (15 animals) was administered as an oral solution of 1 (30 mg / kg) in a saline solution containing 5% NMP (v / v) and 5% solutol HS-15 (v / v) freshly prepared before each administration. The animals were fed with a laboratory rodent diet (Envigo Research Private Ltd, Hyderabad), and animals orally administered with P2 were fasted for 2 h preadministration of the drug and 2 h post-administration of the drug. Water was provided ad libitum. Throughout the experiments, blood samples from Group 1 (iv administered group) were collected immediately before administration of the drug and then at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h and, from Group 2 po administered group) immediately before administration and then at 0.25, 0.5, 1, 2, 4, 8. 12, 24, 36 and 48 h. The blood samples we collected from three mice at each time point and placed in a tube containing K2EDTA anticoagulant and immediately quenched with acetonitrile centrifuged and then stored in the -80 °C freezer for subsequent analysis. Immediately after the collection of the blood, brain samples from three animals at 0.25, 2. 12 and 48 h from each group were collected. The brain samples were homogenized using an ice-cold PBS solution (pH 7.4) in a ratio of 2 buffer:! brain sample. The homogenates were then stored in the - 80 °C freezer for subsequent analysis. The concentration of 1 in the mouse plasma and brain samples was determined using a standard in-house LC-MS / MS analysis. The pharmacokinetic parameters were accessed using a noncompartmental analysis from the Phoenix WinNonlin® (Version 6.3) software. The maximum concentration (Cmax) and the time to reach the maximum concentration (Tmax) were derectly derived from the observed data. The areas under the concentration curve (AUCiast and AUCinf) and the elimination half-life were calculated by the linear trapezoidal rule. The terminal elimination rate constant ke) was determined by regression analysis of the linear terminal portion of the log[P2] vs time curve. The half-life (ti / 2) of P2 in the system was calculated from the equation In 2 / ke. The iv clearance and the steady state volume were calculated, respectively, by the equations CLiv= dose / AUCinf; Vss = MRT x CLiv. The oral bioavailability of P2 was calculated using the formula %F = [(mean AUCpox doseiv) / (mean AUCivx dosepo)] x 100. Tables 4 and 5 show pharmacokinetic studies data of compound 1.

[0257] Table 4. Individual brain concentration-time data and the brain-to-plasma ratio of compound 1 following a single intravenous administration to male C57BL / 6 mice (Dose: 5 mg / kg).

[0258] „... Plasma Brain Brain-to- Mean Time Animal,. „.,,

[0259] . __ concentratio concentration plasma Brain-to-plasma (hr) I® n / (ng / / m »L \) (ng / g) ratio ratio

[0260] 1 245.72 142.35 0.58

[0261] 0.25 2 486.89 130.62 0.27 0.45

[0262] 3 287.81 142.53 0.50

[0263]

[0264] 10 0.00 0.00 NA

[0265] 48 11 0.00 0.00 NA NA

[0266] 12 0.00 0.00 NA

[0267] LLOQ = 30.27 ng / g (Brain); NA - Not applicable

[0268] Table 5. Individual brain concentration-time data and the brain-to-plasma ratio of compound 1 following a single oral administration to male C57BL / 6 mice (Dose: 30 mg / kg).

[0269] ™. Plasma Brain Brain-to- Mean Time Animal,..., „.,, thr) ID concentrate concentration plasma Brain-to-plasma ' ' on (ng / mL) (ng / g) ratio ratio

[0270]

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Claims

CLAIMSI / We claim:

1. A method of treating Alzheimer’s disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof; or a pharmaceutical composition comprising the compound of formula (I) and a pharmaceutically acceptable excipient, carrier, or diluent;whereinR1is H or methylene;R2is halogen;x is 0-4;L is - O- (CH2)n-. -CH2-N+HRA-CH2-, -(CH2)n-,-N(RA)-, or -N(RA)-(CH2)n-; RAis C1-6alkyl; or RAtogether with R1form -(CH2)m-;n is 0-3;m is 1-2;M is a counterion; andy is 0 or 1.

2. The method of claim 1, wherein L is -O-(CH2)n-3. The method of claim 1, wherein L is -O-CH2-4. The method of claim 1, wherein L is -CH2-N+HMe-CH2-5. The method of claim 4, wherein M is H2PO4‘ and y is 1.

6. The method of claim 1, wherein L is -(CH2)n-7. The method of claim 6, wherein n is 2.

8. The method of any one of claims 1-7, wherein R2is -Cl and x is 2.

9. The method of any one of claims 1-8, wherein R2is— 3,5-di chloro or -2,4-di chloro.

10. The method of any one of claims 1-9, wherein R1is H.

11. The method of claim 1, wherein the compound is selected from the group consisting of:or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

12. The method of any one of claims 1-11, wherein the subject has oxidative stress damage induced by amyloid-P aggregation.

13. The method of any one of claims 1-12, wherein the subject has ferroptosis-induced neurodegeneration.

14. The method of claim 13, wherein the ferroptosis-induced neurodegeneration is induced by glutathione peroxidase 4 (GPx4) deficiency.

15. The method of any one of claims 1-14, wherein the compound is administered orally.

16. The method of any one of claims 1-15, wherein the brain concentration and the plasma concentration of the compound in the subject has a ratio of at least 0.3 from about 0.25 h to about 2 h after administration.

17. A method of inhibiting amyloid- aggregation and / or reducing amyloid-aggregation-induced toxicity, the method comprising contacting amyloid with the compound according to any one of claims 1-16.

18. The method of claim 17, wherein amyloid P is contacted with the compound in vitro or ex vivo.

19. The method of claim 17, wherein amyloid P is contacted with the compound in vivo.

20. A compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof:whereinR1is H or methylene;R2is halogen;x is 0-2;L is — (CH2)n—, -N(RA)-, or -N(RB)-(CH2)n-;RAis C1-6alkyl;RBtogether with R1form -CH2-;M is a counterion;y is 0 or 1; andwith the proviso thatwhen L is -N(RB)-(CH2)n- and n is 2, R2is not -3,5-dichloro.

21. The compound of claim 20, wherein L is -(CH2)n-22. The compound of claim 21, wherein n is 2.

23. The compound of any one of claims 20-22, wherein R2is -Cl and x is 2.

24. The compound of claim 23, wherein R2is -3,5-dichloro or -2,4-dichloro.

25. The compound of any one of claims 20-24, wherein R1is H.

26. The compound of claim 20, wherein L is -NMe-27. The compound of claim 20, wherein L is -N(RA)-CH2-, and RAtogether with R1form -CH2-.

28. The compound of claim 20, wherein the compound is selected from the group consisting of:or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

29. A pharmaceutical composition comprising the compound according to any one of claims 20-28 and a pharmaceutically acceptable excipient, carrier, or diluent.

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