Class of inhibitors of α-synuclein aggregates, preparation method therefor, and use thereof

Abstract

Provided in the present invention are a class of inhibitors of α-synuclein aggregates, a preparation method therefor, and use thereof. Specifically, provided are a compound of formula I and a pharmaceutically acceptable salt thereof or a deuterated compound thereof. The compound of the present invention exhibits excellent inhibitory activity against α-synuclein aggregates and can be used for treating neurodegenerative diseases.

Description

A class of inhibitors of α-synuclein aggregates, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to an inhibitor of a class of α-synuclein pathological aggregates, its preparation method, and its uses. Background Technology

[0002] Parkinson's disease (PD) is a disabling neurodegenerative disease, primarily characterized by motor and non-motor symptoms. Statistics show that the global prevalence of PD is 0.3%, second only to Alzheimer's disease among all neurodegenerative diseases. The prevalence increases dramatically with age, reaching over 3% in people over 80 years of age. Currently, more than 6 million people worldwide have been diagnosed with PD. Motor symptoms in PD patients mainly include bradykinesia, resting tremor, and muscle rigidity. Common non-motor symptoms include cognitive impairment, sleep problems, and depression, often accompanied by olfactory dysfunction and constipation.

[0003] The specific pathogenesis of PD is not yet clear. Its pathological markers include the formation of abnormal protein aggregates, Lewy bodies (LB), accompanied by the degeneration or loss of dopaminergic neurons in the substantia nigra and a decrease in striatal dopamine. Aggregated and misfolded α-synuclein is the main component of LB, so PD is classified as a synucleinopathy.

[0004] The aberrant aggregation of α-synuclein forming amyloid deposits is a crucial pathological feature of Parkinson's disease (PD), thus attracting widespread attention and research. α-synuclein is a soluble protein ubiquitous in neurons, composed of 140 amino acid residues. Its N-terminus is rich in lysine and involved in interactions with biological membranes, while its C-terminus is disordered and rich in acidic amino acid sequences, believed to be involved in nuclear localization and interactions with metals and other proteins. The central region between these two is called the non-β-amyloid (NAC) region, containing a highly hydrophobic amino acid sequence and associated with α-synuclein oligomerization and flutter. Currently, clinically applied PD treatments can only improve patient symptoms and cannot stop disease progression or cure the disease.

[0005] In the development of Parkinson's disease (PD), the highly ordered structure of pathological protein aggregates formed by the misfolding of α-synuclein makes them difficult for molecular chaperone proteins to recognize and refold, leading to their continuous proliferation and spread in the brain, ultimately promoting disease progression. Therefore, α-synucleinic diseases are considered a major pathogenic factor in PD and other related synucleinopathies, and a promising clinical therapeutic target for PD. Existing small molecule drug-based intervention strategies for α-synuclein protein aggregates mainly fall into two categories: using small molecule inhibitors to prevent the formation and spread of α-synuclein protein aggregates; and using degrading agents to clear already formed α-synuclein aggregates. Most reported α-synuclein degrading agents are PROTACs; however, these PROTACs have unsatisfactory degradative activity against α-synuclein and are difficult to penetrate the brain. Various small molecule inhibitors targeting α-synuclein, such as NPT200-11, Anle138b, and UBC0599, inhibit the formation of oligomers of α-synuclein monomers in vitro, but their specific mechanisms of action, in vivo efficacy, and effects on the endogenous function of α-synuclein monomers still require further investigation.

[0006] In recent years, with the increasing demand for early diagnosis of neurodegenerative diseases and the advancement of positron emission tomography (PET) technology, various small-molecule probes targeting pathological aggregates of proteins such as Aβ, tau, or α-syn have been discovered. By structurally modifying and derivatizing these existing probes, while maintaining their original binding ability to pathological protein aggregates, they can also recruit other proteins to inhibit the former, potentially leading to a new class of small-molecule inhibitors targeting pathological protein aggregates with novel mechanisms and efficacy, thereby enabling clinical intervention for related neurodegenerative diseases. This patent describes the development of an inhibitor that effectively promotes the recognition of α-syn pathological protein aggregates by molecular chaperone proteins and inhibits their further amplification and spread through structural modification and derivatization of small-molecule probes such as PIB targeting α-syn pathological protein aggregates. This provides a candidate drug with clinical translational potential for the intervention and treatment of diseases related to α-syn pathological protein aggregates, such as PD, MSA, and DLB. Summary of the Invention

[0007] The purpose of this invention is to provide an inhibitor of α-synuclein aggregates with novel structure and excellent activity, as well as its preparation method and uses.

[0008] In a first aspect, the present invention provides a compound of formula I, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof.

[0009] Where X1 is O or S;

[0010] X2 and X3 are independently CH or N;

[0011] R1 is a C1-C8 alkoxy group, a C1-C8 haloalkoxy group, A 4-6 membered heterocyclic group consisting of a C1-C8 alkoxy group substituted with 1, 2, or 3 hydroxyl groups, or a -O-C0-C2 alkylene group containing a heteroatom selected from O, N, and S, wherein the heterocyclic group is optionally substituted with 1, 2, or 3 groups selected from the group consisting of OH, C1-C3 alkyl, and -C1-C3 alkylene-OH;

[0012] R2 is H, halogen, C1-C4 alkyl, or C1-C4 haloalkyl;

[0013] R3 is H, C1-C4 alkyl, or C1-C4 haloalkyl;

[0014] R4 is a C1-C4 alkyl or a C1-C4 haloalkyl;

[0015] Alternatively, R3 and R4 together with the N atoms attached to them can form a 3-6 membered heterocyclic group containing one N heteroatom.

[0016] In another preferred embodiment, R1 is a C1-C4 alkoxy group, a C1-C4 haloalkoxy group, or a C1-C4 haloalkoxy group. One or two hydroxyl-substituted C1-C6 alkoxy groups, -O-4-5-membered heterocyclic groups containing one O heteroatom, -O-C1-C2 alkylene-4-5-membered heterocyclic groups containing one O heteroatom, wherein the heterocyclic group is independently and optionally substituted by one or two groups selected from the group consisting of OH, C1-C2 alkyl and -C1-C2 alkylene-OH.

[0017] In another preferred embodiment, R1 is a C1-C8 alkoxy, a C1-C8 haloalkoxy, or

[0018] In another preferred embodiment, R1 is selected from the group consisting of: methoxy, difluoromethoxy, trifluoromethoxy,

[0019] In another preferred embodiment, X2 is CH and X3 is CH.

[0020] In another preferred embodiment, X2 is CH and X3 is N.

[0021] In another preferred embodiment, X3 is CH and X2 is N.

[0022] In another preferred embodiment, X1 is 0.

[0023] In another preferred example, X1 is S.

[0024] In another preferred embodiment, X1 is O, X2 is CH, and X3 is CH.

[0025] In another preferred embodiment, R1 is a C1-C4 alkoxy, a C1-C4 haloalkoxy, or

[0026] In another preferred embodiment, R1 is methoxy or ethoxy, preferably methoxy.

[0027] In another preferred embodiment, R1 is a halomethoxy group, preferably mono-, di-, or trifluoromethoxy.

[0028] In another preferred embodiment, R1 is

[0029] In another preferred embodiment, R2 is H, a halogen, or a halomethyl group, and preferably R2 is H, F, Cl, Br, or trifluoromethyl.

[0030] In another preferred embodiment, R2 is H.

[0031] In another preferred embodiment, R2 is F.

[0032] In another preferred embodiment, R3 is a C1-C4 alkyl and R4 is a C1-C4 alkyl, preferably R3 is a C1-C2 alkyl and R4 is a C1-C2 alkyl.

[0033] In another preferred embodiment, R3 is H, C1-C4 alkyl and R4 is C1-C4 alkyl, or R3 and R4 together with the N attached thereto form a 3-6 membered heterocyclic group containing one N heteroatom.

[0034] In another preferred embodiment, R3 is a C1-C2 alkyl group and R4 is a C1-C2 alkyl group, or R3 and R4 together with the N attached thereto form a 3-5 membered heterocyclic group containing one N heteroatom.

[0035] In another preferred embodiment, R3 is methyl and R4 is methyl.

[0036] In another preferred embodiment, R3 is H and R4 is methyl.

[0037] In another preferred embodiment, R3 and R4 together with the N attached thereto form a nitrogen-containing heterocyclic butyl group.

[0038] In another preferred embodiment, when X1 is S, R3 and R4, together with the N bonded to them, form a 3-6 membered heterocyclic group containing one N heteroatom, or R1 is...

[0039] In another preferred embodiment, when X1 is 0, R2 and R3 are not simultaneously H.

[0040] In another preferred embodiment, the compound is selected from the group consisting of:

[0041] In another preferred embodiment, X1, X2, X3, R1, R2, R3, and R4 are independently and optionally the corresponding groups in the compounds of the embodiments of this application.

[0042] A second aspect of the present invention provides a pharmaceutical composition comprising:

[0043] a. A pharmaceutically acceptable salt of a compound as described in the first aspect of the invention, or a deuterated compound thereof; and

[0044] b. A pharmaceutically acceptable carrier.

[0045] A third aspect of the invention provides the use of a compound as described in the first aspect of the invention, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a pharmaceutical composition as described in the second aspect of the invention, in the preparation of an α-synuclein aggregate inhibitor.

[0046] In another preferred embodiment, the compound or pharmaceutical composition inhibits the formation of α-synuclein aggregates by inhibiting phosphorylation of serine at position 129 of α-synuclein.

[0047] In another preferred embodiment, the compound or pharmaceutical composition can promote the binding of HSP40 protein to α-syn pff and inhibit fibrous growth.

[0048] A fourth aspect of the invention provides the use of a compound as described in the first aspect of the invention, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a pharmaceutical composition as described in the second aspect of the invention, in the preparation of a medicament for the prevention or treatment of neurodegenerative diseases associated with α-synuclein aggregates or misfolded protein aggregates thereof.

[0049] In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of: Parkinson's disease, multiple system atrophy, Lewy body dementia, Alzheimer's disease, or amyotrophic lateral sclerosis.

[0050] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0051] Figure 1 shows the effect of representative compounds on the phosphorylation of serine at position 129 of α-syn.

[0052] Figure 2 shows that the effect of compound HQY-10-27 is concentration- and time-dependent.

[0053] Figure 3 shows the effect of different concentrations of the compound on the formation of fibrillary aggregates of neurons induced by α-syn pff.

[0054] Figure 4 shows the statistical analysis of the fluorescence experiment.

[0055] Figure 5 shows the volcano plot of the protein spectrum of compound HQY-10-71.

[0056] Figure 6 shows the results of the competitive pulldown experiment.

[0057] Figures 7a, 7b, 7c, and 7d show the kinetic curves of the compound with α-syn pff, HSP40, HSP70, and HSP90 proteins, respectively.

[0058] Figure 8 shows that the compound can promote the binding of HSP40 protein to α-syn pff.

[0059] Figures 9a and 9b show that compound HQY-10-27 can inhibit α-syn fiber growth.

[0060] Figure 10 shows the weight changes in a mouse model of Parkinson's disease.

[0061] Figure 11 shows the changes in the grip strength of the mice.

[0062] Figure 12 shows a pathological image of phosphorylated S129α-syn in the mouse brain region.

[0063] Figure 13 shows the area of ​​pathologically phosphorylated S129α-syn in the mouse brain. Detailed Implementation

[0064] Through extensive and in-depth research, including numerous screenings and tests, the inventors have developed a novel class of inhibitors of α-synuclein aggregates with superior activity, along with their preparation methods and applications. This invention is based on these findings.

[0065] the term

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0067] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”.

[0068] As used herein, the term "alkyl" refers to a monovalent, straight-chain or branched saturated hydrocarbon group consisting only of carbon and hydrogen atoms. For example, "C 1-"C8 alkyl" indicates an alkyl group having 1 to 8 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, or 8). Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. 1- C4 alkyl and C 1- "C2 alkyl" has a similar meaning.

[0069] As used herein, the term "haloalkyl" refers to a group obtained by substituting one or more hydrogen atoms in an alkyl group as described above with the same or different halogens. Wherein, "C..." 1-8 "Halogenated alkyl" is preferably a halogenated C 1-4 Alkyl groups, examples of which include, but are not limited to: -CH2Cl, -CHF2, -CH2CF3, -CH2CCl3, perfluoroalkyl groups (e.g., -CF3-, -CF2CF3), etc.

[0070] As used herein, the term "alkoxy" refers to the formula -OR z Group, wherein R z Alkyl groups are defined herein. Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, isopropoxy, tert-butoxy, -CH2O-CH3, -CH2CH2-O-CH3, -CH2-O-CH2CH3, etc.

[0071] As used herein, the term "haloalkoxy" refers to a group obtained by substituting one or more hydrogen atoms in an alkoxy group as described above with the same or different halogens. Examples of haloalkoxy groups include, but are not limited to, trifluoromethoxy, difluoromethoxy, 2,2,2-trifluoroethoxy, etc.

[0072] As used herein, "halogen" refers to halogens and their isotopes, including but not limited to F, 18 F, 32 Cl, Br, I.

[0073] As used herein, the term "heterocyclic group" refers to a fully or partially saturated monocyclic, bicyclic, or polycyclic cyclic group containing one or more heteroatoms selected from N, S, or O. For example, "3-6 membered heterocyclic group" refers to a group having 3-6 ring members. The nitrogen or sulfur atom may be oxidized, or the nitrogen atom may be quaternized. The heterocyclic group can be attached to any heteroatom or carbon residue in a ring or ring system molecule. Monocyclic heterocyclic groups include, but are not limited to: azacyclic butyl, pyrrolyl, oxacyclic butyl, pyrazolinyl, imidazolinyl, imidazoalkyl, oxazolinyl, isoxazolinyl, thiazoalkyl, isothiazolinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperidinyl, 2-oxopiperidinyl, 2-oxopiperylyl, hexahydroachenginyl, 4-piperidinoneyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxane, and tetrahydro-1,1-dioxothiophene, etc. Polycyclic heterocyclic groups include, but are not limited to, spirocyclic, fused-ring, and bridged-ring heterocyclic groups; wherein the spirocyclic, fused-ring, and bridged-ring heterocyclic groups involved are optionally connected to other groups by single bonds, or further cyclically linked to other cycloalkyl, heterocyclic, aryl, and heteroaryl groups by any two or more atoms on the ring.

[0074] As used herein, the term "substitution" refers to the replacement of one or more hydrogen atoms on a particular group by a particular substituent. The particular substituent is either the substituent described accordingly above or the substituent appearing in the various embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a particular group at any substituted site of that group, and the substituents may be the same or different at each position. Those skilled in the art will understand that the combinations of substituents contemplated in this invention are those that are stable or chemically feasible.

[0075] Unless otherwise specified, the groups described in this invention may be substituted with substituents selected from the group consisting of: D, halogen, cyano, nitro, hydroxyl, amino, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, 3-12 membered heterocyclic groups, C3-C6 alkyl, ... 12 cycloalkyl, 5-10 heteroaryl and C6-C 10 Aryl.

[0076] In this document, “optionally” means that the event or condition described below may, but is not required to, occur, and the description includes both the possibility that the event or condition occurs and the possibility that the event or condition does not occur.

[0077] In this article, the term "multiple" refers to 2, 3, 4, 5, or a positive integer greater than 5.

[0078] As used in this article, the terms “α-synuclein aggregate,” “α-syn aggregate,” and “α-syn filament” are synonymous.

[0079] Active ingredients

[0080] As used herein, “compound of the present invention” means a compound of formula I, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, and also includes its stereoisomers, optical isomers, crystal forms, isotopic derivatives thereof, solvates thereof, or hydrates thereof.

[0081] Unless otherwise specified, the structural formulas described in this invention are intended to include all stereoisomers (such as cis-trans isomers, enantiomers, diastereomers, and conformational isomers): R and S configurations containing an asymmetric center, (Z) and (E) isomers of double bonds, cis-trans isomers of cycloalkanes, etc. Therefore, any single stereochemical isomer of the compounds of this invention, or a mixture of its enantiomers, diastereomers, or conformational isomers, is within the scope of this invention.

[0082] This invention also includes isotopically labeled compounds (i.e., isotopic derivatives), equivalent to the original compounds disclosed herein. However, it is common practice to see one or more atoms replaced by atoms with different atomic weights or mass numbers. Examples of isotopes in the isotopic derivatives of this invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine isotopes, respectively as follows: 2 H, 3 H, 13 C 11 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F and 36 Cl. Isotope derivatives of the compounds of this invention are all within the scope of protection of this invention. In this document, 3H-labeled compounds and 14 C-labeled compounds are useful in tissue distribution experiments of drugs and substrates. Tritium (i.e., 3 H) and carbon- 14 (Right now 14 C) Labeled compounds are relatively easy to prepare and detect, making them the preferred choice among isotopes. Furthermore, heavier isotope substitutions, such as deuterium (2H), are preferred in certain cases due to their excellent metabolic stability, which offers advantages in some therapies, such as increasing half-life or reducing dosage in vivo. Isotope-labeled compounds can be prepared using general methods by replacing non-isotopic reagents with readily available isotope-labeling reagents, as described in the examples.

[0083] As used herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0084] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared using methods known in this field.

[0085] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0086] Metabolites of the compound shown in Formula I and its pharmaceutically acceptable salts, as well as prodrugs that can be converted in vivo into the compound shown in Formula I and its pharmaceutically acceptable salts, are also included within the scope of protection of this invention.

[0087] As used herein, the term "solvent" refers to a complex of a compound of Formula I that coordinates with a solvent molecule in a specific ratio.

[0088] Pharmaceutical Compositions and Administration

[0089] Because the compounds of this invention can inhibit the phosphorylation of α-synuclein S129 or inhibit the elongation of α-syn fibers, thereby inhibiting the formation of α-syn aggregates, and are used for the prevention or treatment of neurodegenerative diseases (such as Parkinson's disease, Lewy body dementia, multiple system atrophy, Alzheimer's disease, amyotrophic lateral sclerosis, etc.) associated with α-synuclein aggregates and their misfolded proteins, the compounds of this invention, their stereoisomers, their optical isomers, their pharmaceutically acceptable salts, their crystal forms, their isotopic derivatives, their prodrugs, their metabolites, their solvates or hydrates thereof, and pharmaceutical compositions containing the compounds of this invention as the main active ingredient, can be used as drugs for the prevention or treatment (stabilization, reduction or cure) of neurodegenerative diseases (such as Parkinson's disease, Lewy body dementia, multiple system atrophy, Alzheimer's disease, amyotrophic lateral sclerosis, etc.) associated with α-synuclein aggregates and their misfolded proteins.

[0090] The pharmaceutical compositions of the present invention comprise the compound of the present invention within a safe and effective range and a pharmaceutically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to treat or diagnose a disease without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-200 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0091] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as Tween). Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0092] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0093] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0094] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0095] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0096] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0097] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0098] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0099] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0100] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0101] The general range of therapeutically effective doses of a compound of Formula I or a composition thereof will be: about 1-2000 mg / day, about 10-about 1000 mg / day, about 10-about 500 mg / day, about 10-about 250 mg / day, about 10-about 100 mg / day, or about 10-about 50 mg / day. The therapeutically effective dose will be administered in one or more doses. However, it should be understood that the specific dose of the compound of the invention for any particular patient will depend on a variety of factors, such as the patient's age, sex, weight, general health condition, diet, individual response, timing of administration, severity of the disease being treated, the activity of the specific compound administered, dosage form, mode of administration, and concomitant medications. The therapeutically effective dose in a given situation can be determined by routine laboratory testing and is within the competence and judgment of a clinician or physician. In any case, the compound or composition will be administered in multiple doses based on the individual patient's condition and in a manner that allows for the delivery of a therapeutically effective dose.

[0102] The main advantages of this invention include:

[0103] The compound presented in this application has a novel structure and can specifically inhibit α-synuclein aggregates. It can be used to prepare drugs for the prevention or treatment of neurodegenerative diseases related to α-synuclein aggregates and their misfolded proteins, which has important clinical significance.

[0104] Animal experiments have shown that the present invention can effectively improve the behavior of mice with Parkinson's disease, and has no obvious toxic side effects, making it very suitable for pharmaceutical development.

[0105] The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0106] Example 1: Synthetic route of compound HQY-7-68

[0107] Synthesis of compound HQY-7-68

[0108] 2-Bromo-6-methoxybenzothiazole (242 mg, 1 mmol), 4-(methylamino)phenylboronic acid (151 mg, 1 mmol), and K2CO3 (207 mg, 1.5 mmol) were dissolved in Dioxane (5 mL), placed in a pressure-resistant bottle, and Pd(dppf)2Cl2 (36.5 mg, 0.05 mmol) was added. The mixture was stirred at 80 °C under a nitrogen atmosphere for 4 h. After the reaction was complete, EA and water were added for extraction, dried over anhydrous sodium sulfate, filtered, and purified by silica gel column chromatography. The PE / EA ratio was 0–30%, yielding compound HQY-7-68 (72 mg, 0.2667 mmol), a yellow solid, with a yield of 26.7%.

[0109] 1 H NMR (400MHz, DMSO) δ7.84–7.69(m,3H),7.61(d,J=1.9Hz,1H),7.05(dd,J=8.9,2.3H z,1H),6.64(d,J=8.6Hz,2H),6.45–6.33(m,1H),3.83(s,3H),2.75(d,J=3.5Hz,3H).

[0110] MS-ESI: m / z calculated for C 15 H 14 N2OS,Exact Mass:270.08,found 271.0[M+H] + .

[0111] Example 2: Synthetic route of compound HQY-8-56

[0112] Synthesis of compound HQY-8-53

[0113] Based on the synthetic method of compound HQY-7-68, compound HQY-8-53 (42 mg, 0.155 mmol), a pale yellow solid, was obtained in 16.4% yield. MS-ESI: m / z calculated for C 13 H9FN2OS,Exact Mass:260.04,found 261.1[M+H] + .

[0114] Synthesis of compound HQY-8-56

[0115] HQY-8-53 (18 mg, 0.07 mmol) was dissolved in EtOH (1 mL), and dimethylamine (6.2 mg, 0.14 mmol) was added. The mixture was stirred overnight at 80 °C. After the reaction was complete, the solvent was removed by vacuum distillation, and the mixture was purified by silica gel column chromatography (PE / EA 0–80%). The purified mixture was then purified by HPLC and lyophilized to give compound HQY-8-56 (2.1 mg, 0.0074 mmol), a white solid with a yield of 10.5%.

[0116] 1 H NMR (400MHz, DMSO) δ8.62(d,J=1.3Hz,1H),8.22(d,J=9.3Hz,1H),7.89(d,J=8.9Hz,1H),7.70( d,J=2.0Hz,1H),7.12(dd,J=8.8,2.0Hz,1H),7.01(d,J=9.2Hz,1H),3.85(s,3H),3.20(s,6H).

[0117] MS-ESI: m / z calculated for C 15 H 15 N3OS,Exact Mass:285.09,found 286.1[M+H] + .

[0118] Example 3: Synthetic route of compound HQY-8-61

[0119] Synthesis of compound HQY-8-61

[0120] 2-Amino-4-methoxyphenol (46 mg, 0.33 mmol) and N-methyl-4-(trifluoromethyl)aniline (50 mg, 0.33 mmol) were mixed in a flask, and sodium hydroxide aqueous solution (1 M, 5 mL) was added. The mixture was stirred at 90 °C for 2 h. After the reaction was complete, EA and water were added for extraction. The mixture was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The mixture was then purified by HPLC and lyophilized to give compound HQY-8-61 (2.2 mg, 0.0087 mmol), a pale yellow solid with a yield of 2.6%.

[0121] 1 H NMR (400MHz, DMSO) δ7.90(d,J=8.6Hz,2H),7.56(d,J=8.8Hz,1H),7.22(d,J=2.1Hz ,1H),6.88(dd,J=8.8,2.1Hz,1H),6.68(d,J=8.6Hz,2H),3.80(s,3H),2.76(s,3H).

[0122] MS-ESI: m / z calculated for C 15 H 14 N2O2, Exact Mass:254.11, found 255.1[M+H] + .

[0123] Example 4: Synthetic route of compound HQY-8-77

[0124] Synthesis of compound HQY-8-77

[0125] 2-Amino-4-fluorophenol (42 mg, 0.33 mmol) and 4-methylaminobenzoic acid (50 mg, 0.33 mmol) were mixed in a flask, and PPA (1 g) was added. The mixture was stirred at 180 °C for 30 min. After the reaction was complete, it was cooled to room temperature, quenched with potassium carbonate aqueous solution, extracted with DCM and water, dried over anhydrous sodium sulfate, filtered, purified by silica gel column chromatography (PE / EA 0–50%), purified by HPLC, and lyophilized to give compound HQY-8-77 (7.3 mg, 0.0302 mmol), a white solid with a yield of 9.1%.

[0126] 1H NMR (400MHz, DMSO) δ7.92(d,J=8.4Hz,2H),7.69(dd,J=8.7,4.4Hz,1H),7.52(d,J=8.9Hz,1H ),7.14(t,J=9.4Hz,1H),6.69(d,J=8.5Hz,2H),6.62(d,J=4.1Hz,1H),2.77(d,J=4.2Hz,3H).

[0127] MS-ESI: m / z calculated for C 14 H 11 FN2O,Exact Mass:242.09,found 243.1[M+H] + .

[0128] Synthetic route of compound HQY-10-27 in Example 5

[0129] Synthesis of compound HQY-10-27

[0130] 2-Amino-5-methoxyphenol (139 mg, 1 mmol) and 4-(dimethylamino)benzaldehyde (189 mg, 1 mmol) were dissolved in MeOH (5 mL), and TEA (310 μL, 2.4 mmol) was added. The mixture was stirred at room temperature for 30 min, and then DDQ (227 mg, 1.00 mmol) and DCM (5 mL) were added. The mixture was stirred at room temperature for another 30 min. After the reaction was complete, the solvent was removed by vacuum distillation. The mixture was extracted with DCM, water, and a saturated sodium bicarbonate aqueous solution. After drying with anhydrous sodium sulfate, the mixture was filtered and purified by silica gel column chromatography (PE / EA 0–60%) to give compound HQY-10-27 (128 mg, 0.4776 mmol), a grayish-white solid with a yield of 47.8%.

[0131] 1 H NMR (400MHz, DMSO) δ7.94(d,J=8.5Hz,2H),7.56(d,J=8.6Hz,1H),7.34(s,1H),6.93(d,J=8.8Hz,1H),6.85(d,J=8.7Hz,2H),3.82(s,3H),3.03(s,6H).

[0132] MS-ESI: m / z calculated for C 16 H 16 N2O2, Exact Mass:268.12, found 269.1[M+H] + .

[0133] Synthetic route of compound HQY-10-29 in Example 6

[0134] Synthesis of compound HQY-10-21

[0135] HQY-9-64 (137 mg, 0.482 mmol) was dissolved in DCM (5 mL). A solution of boron tribromide in dichloromethane (1 M, 964 μL) was slowly added under a nitrogen atmosphere at -78 °C. The mixture was stirred overnight at room temperature, then heated to 35 °C and stirred for another 3 hours. After the reaction was complete, a small amount of water was added to quench the reaction. The solution was then processed using DCM and... i Extracted with a PrOH / CCl3H (1:3) mixed solution, dried over anhydrous sodium sulfate, filtered, and purified by silica gel column chromatography (DCM / MeOH, 0–20%), yielding compound HQY-10-21 (112 mg, 0.4148 mmol) as a yellow solid, in 86.1% yield. MS-ESI: m / z calculated for C 15 H 14 N2OS,Exact Mass:270.08,found 271.1[M+H] + .

[0136] Synthesis of compound HQY-10-26

[0137] HQY-10-21 (27 mg, 0.1 mmol) and 1,2-dibromoethane (31 mg, 0.12 mmol) were dissolved in MeCN (1 mL), and Cs₂CO₃ (65 mg, 0.2 mmol) was added. The mixture was stirred overnight at 70 °C. After the reaction was complete, the mixture was filtered and purified by silica gel column chromatography (DCM / EA 0–100%) to give compound HQY-10-26 (32 mg, 0.0851 mmol), a gray solid, with a yield of 85.1%. MS-ESI: m / z calculated for C 17 H 17 BrN2OS,Exact Mass:376.02,found 377.1[M+H] + .

[0138] Synthesis of compound HQY-10-29

[0139] HQY-10-26 (5 mg, 0.016 mmol) and N-methylpiperazine (3.2 mg, 0.032 mmol) were dissolved in MeCN (0.2 mL), and K2CO3 (6.6 mg, 0.048 mmol) was added. The mixture was stirred at 70 °C for 1 h. After the reaction was complete, the mixture was purified by HPLC and lyophilized to obtain compound HQY-10-29 (1.3 mg, 0.0037 mmol), a yellow solid, with a yield of 23.1%.

[0140] 1 H NMR (400MHz, DMSO) δ7.83(d,J=8.7Hz,3H),7.66(d,1H),7.08(dd,J=8.9,2.2Hz,1H),6.81(d,J=8.9Hz,2H ),4.26–4.20(m,2H),3.42–3.38(m,2H),3.40–3.19(m,6H),3.15–3.06(m,2H),3.01(s,6H),2.78(s,3H).

[0141] MS-ESI: m / z calculated for C 22 H 28 N4OS,Exact Mass:396.20,found 397.1[M+H] + .

[0142] Synthesis of compound HQY-8-2

[0143] Replace the starting material 4-(methylamino)phenylboronic acid with 2-methylamino-5-pyridineboronic acid; refer to the other reaction steps.

[0144] Example 1 yielded compound HQY-8-2.

[0145] 1 H NMR (400MHz, DMSO) δ8.59(d,J=1.0Hz,1H),8.10(d,J=9.0Hz,1H),7.86(d,J=8.9Hz,1H),7.85(s,1H) ,7.68(d,J=1.9Hz,1H),7.10(dd,J=8.9,1.8Hz,1H),6.75(d,J=9.0Hz,1H),3.84(s,3H),2.90(s,3H).

[0146] MS-ESI: m / z calculated for C 14 H 13N3OS,Exact Mass:271.08,found 272.0[M+H] + .

[0147] Synthesis of compound HQY-8-71

[0148] The starting material 2-bromo-6-methoxybenzothiazole was replaced with 2-bromo-6-fluorobenzothiazole, and the starting material 4-(methylamino)phenylboronic acid was replaced with 2-methylamino-5-pyridineboronic acid. Other reaction steps were performed as described in Example 1 to obtain compound HQY-8-71.

[0149] 1 H NMR (400MHz, DMSO) δ8.63(s,1H),8.14(d,J=8.7Hz,1H),8.04(dd,J=8.7,2.5Hz,1H),7.9 9(dd,J=8.9,5.0Hz,1H),7.38(td,J=9.1,2.7Hz,1H),6.79(d,J=8.7Hz,1H),2.92(s,3H).

[0150] MS-ESI: m / z calculated for C 13 H 10 FN3S,Exact Mass:259.06,found 260.1[M+H] + .

[0151] Synthesis of compound HQY-8-74

[0152] The starting material 2-bromo-6-methoxybenzothiazole was replaced with 2-bromo-6-fluorobenzothiazole, and the other reaction steps were the same as in Example 1, to obtain compound HQY-8-74.

[0153] 1 H NMR (400MHz, DMSO) δ7.95 (dd, J=8.7, 2.5Hz, 1H), 7.91 (dd, J=8.9, 4.9Hz, 1H), 7.80 (d,J=8.7Hz,2H),7.32(td,J=9.1,2.6Hz,1H),6.66(d,J=8.7Hz,2H),2.76(s,3H).

[0154] MS-ESI: m / z calculated for C 14 H 11 FN2S,Exact Mass:258.06,found 259.1[M+H] + .

[0155] Synthesis of compound HQY-8-78

[0156] The starting material 2-bromo-6-methoxybenzothiazole was replaced with 2-bromo-6-chlorobenzothiazole, and the starting material 4-(methylamino)phenylboronic acid was replaced with 2-methylamino-5-pyridineboronic acid. Other reaction steps were performed as described in Example 1 to obtain compound HQY-8-78.

[0157] 1 H NMR (400MHz, DMSO) δ8.67(s,1H),8.26(s,1H),8.10(d,J=8.9Hz,1H),7.95(b,J=8 .6Hz,1H),7.75(s,1H),7.53(d,J=8.7Hz,1H),6.72(d,J=8.6Hz,1H),2.90(s,3H).

[0158] MS-ESI: m / z calculated for C 13 H 10 ClN3S,Exact Mass:275.03,found 276.0[M+H] + .

[0159] Synthesis of compound HQY-8-82

[0160] The starting material 2-bromo-6-methoxybenzothiazole was replaced with 2-bromo-6-chlorobenzothiazole, and the other reaction steps were the same as in Example 1, to obtain compound HQY-8-82.

[0161] 1 H NMR (400MHz, DMSO) δ8.18(s,1H),7.88(d,J=8.7Hz,1H),7.81(d,J=8.4Hz,2H),7.48( d,J=9.3Hz,1H),6.65(d,J=8.5Hz,2H),6.54(d,J=4.5Hz,1H),2.76(d,J=4.8Hz,3H).

[0162] MS-ESI: m / z calculated for C 14 H 11 ClN2S,Exact Mass:274.03,found 275.0[M+H] + .

[0163] Synthesis of compound HQY-9-8

[0164] The starting material 2-bromo-6-methoxybenzothiazole was replaced with 2-bromo-6-cyanobenzothiazole, and the starting material 4-(methylamino)phenylboronic acid was replaced with 2-methylamino-5-pyridineboronic acid. Other reaction steps were performed as described in Example 1 to obtain compound HQY-9-8.

[0165] 1 H NMR (400MHz, DMSO) δ8.76 (d, J=2.2 Hz,1H),8.69(d,J=1.3Hz,1H),8.08(d,J=8.5Hz,2H),7.89(dd,J=8.4,1.5Hz,1H),6.66(d,J=8.9Hz,1H),2.89(s,3H).

[0166] MS-ESI: m / z calculated for C 14 H 10 N4S,Exact Mass:266.06,found 267.1[M+H] + .

[0167] Synthesis of compound HQY-9-21

[0168] The starting material 4-(methylamino)phenylboronic acid was replaced with 4-methoxyphenylboronic acid, and the other reaction steps were the same as in Example 1, to obtain compound HQY-9-21.

[0169] 1 H NMR (400MHz, DMSO) δ8.83(d,J=1.9Hz,1H),8.31(dd,J=8.7,2.4Hz,1H),7.94(d,J=8.9Hz,1H),7. 73(d,J=2.3Hz,1H),7.14(dd,J=8.9,2.5Hz,1H),7.01(d,J=8.7Hz,1H),3.95(s,3H),3.86(s,3H).

[0170] MS-ESI: m / z calculated for C 14 H 12 N2O2S,Exact Mass:272.06,found 273.0[M+H] + .

[0171] Synthesis of compound HQY-9-22

[0172] The raw material 4-(methylamino)phenylboronic acid was replaced with 4-fluoro-phenylboronic acid, and the other reaction steps were the same as in Example 1, to obtain compound HQY-9-22.

[0173] 1 H NMR (400MHz, DMSO) δ8.10 (dd, J=8.4, 5.5Hz, 2H), 7.94 (d, J=8.9Hz, 1H), 7.73 (d, J=2.2 Hz, 1H), 7.40 (t, J=8.7Hz, 2H), 7.14 (dd, J=8.9, 2.3Hz, 1H), 3.86 (s, 3H).

[0174] MS-ESI: m / z calculated for C 14 H 10 FNOS,Exact Mass:259.05,found 259.89[M+H] + .

[0175] Synthesis of compound HQY-9-23

[0176] Replace the starting material 4-(methylamino)phenylboronic acid with 4-methoxy-phenylboronic acid; refer to the other reaction steps.

[0177] Example 1 yielded compound HQY-9-23.

[0178] 1 H NMR (400MHz, DMSO) δ7.98(d,J=8.8Hz,2H),7.89(d,J=8.9Hz,1H),7.69(d,J=2.4Hz,1H),7.14–7.08(m,3H),3.85(s,6H).

[0179] MS-ESI: m / z calculated for C 15 H 13 N2OS,Exact Mass:271.07,found 272.1[M+H] + .

[0180] Synthesis of compound HQY-9-27

[0181] The raw material 4-(methylamino)phenylboronic acid was replaced with 4-chloro-phenylboronic acid, and the other reaction steps were the same as in Example 1, to obtain compound HQY-9-27.

[0182] 1H NMR (400MHz, DMSO) δ8.06(d,J=8.6Hz,2H),7.96(d,J=8.9Hz,1H),7.75(d,J=2.4Hz,1H),7.62(d,J=8.6Hz,2H),7.15(dd,J=8.9,2.6Hz,1H),3.86(s,3H).

[0183] MS-ESI: m / z calculated for C 14 H 10 ClNOS,Exact Mass:275.02,found 276.0[M+H] + .

[0184] Synthesis of compound HQY-9-64

[0185] The raw material 4-(methylamino)phenylboronic acid was replaced with 4-dimethylaminophenylboronic acid, and the other reaction steps were the same as in Example 1, to obtain compound HQY-9-64.

[0186] 1 H NMR (400MHz, DMSO) δ7.84(d,J=7.3Hz,2H),7.81(d,J=7.3Hz,1H),7.63(d,J=2.5Hz ,1H),7.06(dd,J=8.9,2.6Hz,1H),6.81(d,J=8.9Hz,2H),3.83(s,3H),3.01(s,6H).

[0187] MS-ESI: m / z calculated for C 16 H 16 N2OS,Exact Mass:284.10,found 285.1[M+H] + .

[0188] Synthesis of compound HQY-9-67

[0189] Replacing the starting material 4-(methylamino)phenylboronic acid with 2-fluoro-4-pyridineboronic acid, and following other reaction steps as in Example 1, yielded compound HQY-9-65 (42 mg, 0.155 mmol). Replacing the starting material dimethylamine with methylamine methanol solution (pale yellow solid), and following other reaction steps as in Example 2, yielded compound HQY-9-67.

[0190] 1H NMR (400MHz, CDCl3) δ10.24(s,1H),7.98(d,J=9.0Hz,1H),7.74(d,J=6.4Hz,1H),7 .32(d,J=8.6Hz,2H),7.21(s,1H),7.13(d,J=8.9Hz,1H),3.87(s,3H),3.05(s,3H).

[0191] MS-ESI: m / z calculated for C 14 H 13 N3OS,Exact Mass:271.08,found 272.1[M+H] + .

[0192] Synthesis of compound HQY-9-92

[0193] The raw material 4-(methylamino)phenylboronic acid was replaced with 3-fluoro-4-(methylamino)phenylboronic acid, and the other reaction steps were the same as in Example 1, to obtain compound HQY-9-92.

[0194] 1 H NMR(400MHz,DMSO)δ7.83(d,J=8.9Hz,1H),7.66(d,J=8.5Hz,1H),7.65–7.63(m,1H),7.0 8(dd,J=8.9,2.5Hz,1H),6.76(t,J=8.8Hz,1H),6.30(s,1H),3.84(s,3H),2.80(d,J=4.2 Hz,3H).

[0195] MS-ESI: m / z calculated for C 15 H 13 FN2OS,Exact Mass:288.07,found 289.1[M+H] + .

[0196] Synthesis of compound HQY-8-57

[0197] The dimethylamine raw material was replaced with aziridine, and the other reaction steps were the same as in Example 2, to obtain compound HQY-8-57.

[0198] 1H NMR (400MHz, DMSO) δ8.57 (s, 1H), 8.27 (d, J = 9.2 Hz,1H),7.90(d,J=8.9Hz,1H),7.71(d,J=1.9Hz,1H),7.13(dd,J=8.8,2.0Hz,1H),6. 74(d,J=9.0Hz,1H),4.22(t,J=7.5Hz,4H),3.85(s,3H),2.43(dt,J=14.9,7.4Hz,2H).

[0199] MS-ESI: m / z calculated for C 16 H 15 N3OS,Exact Mass:297.09,found 298.1[M+H] + .

[0200] Synthesis of compound HQY-9-33

[0201] By replacing the dimethylamine with sodium hydroxide and following the other reaction steps as described in Example 2, compound HQY-9-33 was obtained.

[0202] 1 H NMR (400MHz, DMSO) δ12.14(s,1H),8.09(s,1H),8.05(dd,J=9.6,2.5Hz,1H),7.85(d,J=8.9Hz ,1H),7.68(d,J=2.4Hz,1H),7.10(dd,J=8.9,2.5Hz,1H),6.50(d,J=9.6Hz,1H),3.84(s,3H).

[0203] MS-ESI: m / z calculated for C 13 H 10 N2O2S,Exact Mass:258.05,found 259.1[M+H] + .

[0204] Synthesis of compound HQY-9-32

[0205] By replacing dimethylamine with an aqueous solution of ammonia and following the other reaction steps as described in Example 2, compound HQY-9-32 was obtained.

[0206] 1H NMR (400MHz, DMSO) δ8.60(s,1H),8.24(d,J=9.1Hz,1H),7.89(d,J=8.9Hz,1H),7.72(s,1H),7.12(d,J=9.0Hz,1H),6.86(d,J=8.8Hz,1H),3.85(s,3H).

[0207] MS-ESI: m / z calculated for C 13 H 11 N3OS,Exact Mass:257.06,found 258.1[M+H] + .

[0208] Synthesis of compound HQY-8-73

[0209] By replacing N-methyl-4-(trifluoromethyl)aniline with N-methyl-5-(trifluoromethyl)pyridin-2-amine and following other reaction steps as described in Example 3, compound HQY-8-73 was obtained.

[0210] 1 H NMR (400MHz, DMSO) δ8.74(s,1H),8.12(d,J=8.8Hz,1H),7.78(b,1H),7.61(d,J=8.8Hz,1 H),7.27(s,1H),6.94(d,J=6.9Hz,1H),6.73(d,J=8.9Hz,1H),3.81(s,3H),2.90(s,3H).

[0211] MS-ESI: m / z calculated for C 14 H 13 N3O2, Exact Mass:255.10, found 256.1[M+H] + .

[0212] Synthesis of compound HQY-9-56

[0213] By replacing N-methyl-4-(trifluoromethyl)aniline with 2-(azacyclobutane-1-yl)-5-(trifluoromethyl)pyridine, and following other reaction steps as described in Example 3, compound HQY-9-56 was obtained.

[0214] 1H NMR (400MHz, DMSO) δ8.80(d,J=1.8Hz,1H),8.17(dd,J=8.9,2.2Hz,1H),7.61(d,J=8.9Hz,1H),7.27(d,J=2.4Hz,1H), 6.93(dd,J=8.9,2.5Hz,1H),6.53(d,J=8.8Hz,1H),4.11(t,J=7.5Hz,4H),3.81(s,3H),2.39(dt,J=14.9,7.4Hz,2H).

[0215] MS-ESI: m / z calculated for C 16 H 15 N3O2, Exact Mass:281.12, found 282.1[M+H] + .

[0216] Synthesis of compound HQY-11-57

[0217] The raw material 2-amino-4-methoxyphenol was replaced with 2-amino-5-methoxyphenol, and the raw material N-methyl-4-(trifluoromethyl)aniline was replaced with N-methyl-5-(trifluoromethyl)pyridine-2-amine. Other reaction steps were as described in Example 3, and compound HQY-11-57 was obtained.

[0218] 1 H NMR (400MHz, DMSO) δ8.71(d,J=1.7Hz,1H),8.09(d,J=7.7Hz,1H),7.70(s,1H),7.60(d,J=8.7Hz,1H) ,7.36(d,J=2.2Hz,1H),6.96(dd,J=8.7,2.3Hz,1H),6.72(d,J=8.9Hz,1H),3.83(s,3H),2.90(s,3H).

[0219] MS-ESI: m / z calculated for C 14 H 13 N3O2, Exact Mass:255.10, found 256.1[M+H] + .

[0220] Synthesis of compound HQY-11-75

[0221] The raw material 2-amino-4-methoxyphenol was replaced with 2-amino-5-methoxyphenol, and the raw material N-methyl-4-(trifluoromethyl)aniline was replaced with 2-dimethylamino-5-(trifluoromethyl)pyridine. Other reaction steps were as described in Example 3, and compound HQY-11-75 was obtained.

[0222] 1 H NMR (400MHz, DMSO) δ8.81(s,1H),8.13(d,J=7.8Hz,1H),7.59(d,J=7.9Hz,1H),7. 36(s,1H),6.96(d,J=5.9Hz,1H),6.83(d,J=7.7Hz,1H),3.83(s,3H),3.14(s,6H).

[0223] MS-ESI: m / z calculated for C 15 H 15 N3O2, Exact Mass:269.12, found 270.03[M+H] + .

[0224] Synthesis of compound HQY-12-2

[0225] The raw material 2-amino-4-methoxyphenol was replaced with 2-amino-5-methoxyphenol, and the raw material N-methyl-4-(trifluoromethyl)aniline was replaced with 2-(azacyclobutane-1-yl)-5-(trifluoromethyl)pyridine. Other reaction steps were as described in Example 3, and compound HQY-12-2 was obtained.

[0226] 1 H NMR (400MHz, DMSO) δ8.76(d,J=1.6Hz,1H),8.16(dd,J=8.9,2.2Hz,1H),7.60(d,J=8.7Hz,1H),7.37(d,J=2.2Hz,1H), 6.97(dd,J=8.7,2.3Hz,1H),6.57(d,J=8.9Hz,1H),4.12(t,J=7.5Hz,4H),3.83(s,3H),2.40(dd,J=15.0,7.6Hz,2H).

[0227] MS-ESI: m / z calculated for C 16 H 15 N3O2, Exact Mass:281.12, found 282.1[M+H] + .

[0228] Synthesis of compound HQY-12-13

[0229] The raw material 2-amino-4-methoxyphenol was replaced with 2-amino-5-methoxyphenol, and the raw material N-methyl-4-(trifluoromethyl)aniline was replaced with 2-methoxy-5-trifluoromethylpyridine. Other reaction steps were as described in Example 3, and compound HQY-12-13 was obtained.

[0230] 1 H NMR (400MHz, DMSO) δ8.93(d,J=2.4Hz,1H),8.37(dd,J=8.8,2.4Hz,1H),7.68(d,J=8.8Hz,1H),7. 42(d,J=2.3Hz,1H),7.05(d,J=8.7Hz,1H),7.01(dd,J=8.9,2.2Hz,1H),3.96(s,3H),3.85(s,3H).

[0231] MS-ESI:m / z calculated for Chemical Formula:C 14 H 12 N2O3, Exact Mass:256.08, found 257.1[M+H] + .

[0232] Synthesis of compound HQY-12-15

[0233] The raw material 2-amino-4-methoxyphenol was replaced with 2-amino-5-methoxyphenol, and the raw material N-methyl-4-(trifluoromethyl)aniline was replaced with N-methyl-4-(trifluoromethyl)pyridine-2-amine. Other reaction steps were as described in Example 3, and the compound HQY-12-15 was obtained.

[0234] 1 H NMR (400MHz, DMSO) δ8.28(d,J=5.4Hz,1H),7.75(d,J=9.0Hz,1H),7.42(d,J=2.6Hz,1H),7 .36(s,1H),7.29(d,J=6.0Hz,1H),7.10(dd,J=9.0,2.6Hz,1H),3.84(s,3H),3.17(s,6H).

[0235] MS-ESI:m / z calculated for Chemical Formula:C 14 H 13N3O2, Exact Mass:255.10, found 256.2[M+H] + .

[0236] Synthesis of compound HQY-12-16

[0237] The raw material 2-amino-4-methoxyphenol was replaced with 2-amino-5-methoxyphenol, and the raw material N-methyl-4-(trifluoromethyl)aniline was replaced with 2-(azacyclobutane-1-yl)-4-(trifluoromethyl)pyridine. Other reaction steps were as described in Example 3, and compound HQY-12-16 was obtained.

[0238] 1 H NMR (400MHz, DMSO) δ8.22(d,J=5.8Hz,1H),7.76(d,J=9.0Hz,1H),7.42(d,J=2.6Hz,1H),7.3 3(d,J=5.7Hz,1H),7.17–7.07(m,2H),4.17(t,J=7.5Hz,4H),3.84(s,3H),2.46–2.36(m,2H).

[0239] MS-ESI:m / z calculated for Chemical Formula:C 16 H 15 N3O2, Exact Mass:281.12, found 282.1[M+H] + .

[0240] Synthesis of compound HQY-12-18

[0241] The raw material 2-amino-4-methoxyphenol was replaced with 2-amino-5-methoxyphenol, and the raw material N-methyl-4-(trifluoromethyl)aniline was replaced with 2-methoxy-5-trifluoromethylpyridine. Other reaction steps were as described in Example 3, and compound HQY-12-18 was obtained.

[0242] 1 H NMR (400MHz, DMSO) δ8.96(d,J=2.1Hz,1H),8.40(dd,J=8.7,2.4Hz,1H),7.68(d,J=8.5Hz,1H ),7.36(s,1H),7.06(d,J=8.7Hz,1H),7.00(dd,J=8.9,2.5Hz,1H),3.97(s,3H),3.83(s,3H).

[0243] MS-ESI:m / z calculated for Chemical Formula:C 14 H 12 N2O3, Exact Mass:256.08, found 257.2[M+H] + .

[0244] Synthesis of compound HQY-8-76

[0245] By replacing 4-methylaminobenzoic acid with 6-methylaminonicotinic acid and following other reaction steps as described in Example 4, compound HQY-8-76 was obtained.

[0246] 1 H NMR (400MHz, DMSO) δ8.78(d,J=1.7Hz,1H),8.12(d,J=8.8Hz,1H),7.75(dd,J=8.9,4.4Hz,1H) ,7.59(dd,J=8.8,2.5Hz,1H),7.21(td,J=9.7,2.5Hz,1H),6.72(d,J=8.9Hz,1H),2.90(s,3H).

[0247] MS-ESI: m / z calculated for C 13 H 10 FN3O,Exact Mass:243.08,found 243.9[M+H] + .

[0248] Synthesis of compound HQY-9-62

[0249] By replacing 4-methylaminobenzoic acid with 6-(azacyclobutan-1-yl)nicotinic acid and following other reaction steps as described in Example 4, compound HQY-9-62 was obtained.

[0250] 1 H NMR (400MHz, DMSO) δ8.89 (s, 1H), 8.34 (d, J = 9.2 Hz,1H),7.83(dd,J=8.8,4.3Hz,1H),7.72(d,J=6.8Hz,1H),7.35(d,J=7.3Hz,1H) ,7.14(d,J=9.4Hz,1H),4.48–4.38(m,2H),3.55–3.48(m,2H),2.20–2.11(m,2H).

[0251] MS-ESI: m / z calculated for C15 H 12 FN3O,Exact Mass:269.10,found 270.1[M+H] + .

[0252] Synthesis of compound HQY-12-105

[0253] The raw material 2-amino-4-fluorophenol was replaced with 2-amino-5-trifluoromethylphenol, and the other reaction steps were the same as in Example 4, to obtain compound HQY-12-105.

[0254] 1 H NMR (400MHz, DMSO) δ8.13(s,1H),7.96(d,J=8.8Hz,2H),7.85(d,J=8.3Hz,1H) ,7.68(d,J=8.3Hz,1H),6.82–6.72(m,1H),6.71(d,J=8.8Hz,2H),2.78(s,3H).

[0255] MS-ESI: m / z calculated for C 15 H 11 F3N2O,Exact Mass:292.08,found 292.92[M+H] + .

[0256] Synthesis of compound HQY-13-5

[0257] The raw material 2-amino-4-fluorophenol was replaced with 2-amino-5-(methanesulfonyl)phenol, and the other reaction steps were the same as in Example 4, to obtain compound HQY-13-5.

[0258] 1 H NMR (400MHz, DMSO) δ8.22(s,1H),7.98(d,J=8.4Hz,2H),7.88(s,2H),6.72(d,J=8.3Hz,2H),3.28(s,3H),2.79(s,3H).

[0259] MS-ESI: m / z calculated for C 15 H 14 N2O3S,Exact Mass:302.07,found 302.9[M+H] + .

[0260] Synthesis of compound HQY-9-97

[0261] The raw material 4-(dimethylamino)benzaldehyde was replaced with 4-methylaminobenzaldehyde, and the other reaction steps were the same as in Example 5, to obtain compound HQY-9-97.

[0262] 1 H NMR (400MHz, DMSO) δ7.86(d,J=8.6Hz,2H),7.54(d,J=8.7Hz,1H),7.32(d,J=2.1Hz ,1H),6.92(dd,J=8.7,2.1Hz,1H),6.67(d,J=8.7Hz,2H),3.82(s,3H),2.76(s,3H).

[0263] MS-ESI: m / z calculated for C 15 H 14 N2O2, Exact Mass:254.11, found 255.1[M+H] + .

[0264] Synthesis of compound HQY-10-36

[0265] The raw material 2-amino-5-methoxyphenol was replaced with 2-amino-5-fluoro-3-methoxyphenol, and the raw material 4-(dimethylamino)benzaldehyde was replaced with 4-methylaminobenzaldehyde. Other reaction steps were performed as described in Example 5, and compound HQY-10-36 was obtained.

[0266] 1 H NMR (400MHz, DMSO) δ7.86(d,J=8.6Hz,2H),7.59(dd,J=13.9,9.2Hz,2H),6.67(d,J=8.7Hz,2H),6.54(d,J=4.3Hz,1H),3.90(s,3H),2.76(d,J=4.3Hz,3H).

[0267] MS-ESI: m / z calculated for C 15 H 13 FN2O2,Exact Mass:272.10,found 273.0[M+H] + .

[0268] Synthesis of compound HQY-10-100

[0269] The raw material 4-(dimethylamino)benzaldehyde was replaced with 4-(azacyclobutane-1-yl)benzaldehyde, and the other reaction steps were the same as in Example 5, to obtain compound HQY-10-100.

[0270] 1 H NMR (400MHz, DMSO) δ7.85(d,J=8.4Hz,2H),7.54(d,J=8.7Hz,1H),7.32(s,1H),6.92(d,J=8.6Hz,1H),6.71(d,J=8.5Hz,3 H),5.90(ddd,J=15.3,10.3,5.0Hz,1H),5.25(d,J=17.3Hz,1H),5.14(d,J=10.4Hz,1H),3.82(s,3H),3.81–3.75(m,2H).

[0271] MS-ESI: m / z calculated for C 17 H 16 N2O2, Exact Mass:280.12, found 280.99[M+H] + .

[0272] Synthesis of compound HQY-11-51

[0273] The raw material 2-amino-5-methoxyphenol was replaced with 2-amino-5-fluoro-3-methoxyphenol, and the other reaction steps were the same as in Example 5, to obtain compound HQY-11-51.

[0274] 1 H NMR (400MHz, DMSO) δ7.93(d,J=8.4Hz,2H),7.62(d,J=7.6Hz,1H),7.59(d,J=11.4Hz,1H),6.85(d,J=8.5Hz,2H),3.91(s,2H),3.03(s,6H).

[0275] MS-ESI: m / z calculated for C 16 H 15 FN2O2, Exact Mass:286.11, found 287.01[M+H] + .

[0276] Synthesis of compound HQY-10-30

[0277] By replacing the raw material N-methylpiperazine with morpholine, and following the other reaction steps as described in Example 6, compound HQY-10-30 was obtained.

[0278] 1 H NMR (400MHz, DMSO) δ7.90–7.82(m,J=9.3Hz,3H),7.73(d,J=2.4Hz,1H),7.15(dd,J=8.9,2.5Hz,1H),6.82(d,J=9.0Hz,2H ),4.46–4.41(m,2H),4.03–3.98(m,2H),3.75–3.62(m,J=37.6Hz,4H),3.59–3.50(m,2H),3.28–3.18(m,2H),3.02(s,6H).

[0279] MS-ESI: m / z calculated for C 21 H 25 N3O2S,Exact Mass:383.17,found 384.1[M+H] + .

[0280] Synthesis of compound HQY-10-37

[0281] By replacing the starting material N-methylpiperazine with diethylamine and following other reaction steps as described in Example 6, compound HQY-10-37 was obtained.

[0282] 1 H NMR (400MHz, DMSO) δ7.88(s,1H),7.84(d,J=8.8Hz,2H),7.72(s,1H),7.14(d,J=8.8Hz,1H ),6.82(d,J=8.7Hz,2H),4.44–4.33(m,2H),3.59–3.55(m,2H),3.02(s,6H),2.89(s,6H).

[0283] MS-ESI: m / z calculated for C 19 H 23 N3OS,Exact Mass:341.16,found 342.1[M+H] + .

[0284] Synthesis of compound HQY-10-69

[0285] By replacing the raw material 1,2-dibromoethane with the photocrosslinking intermediate PC1, and following the other reaction steps as described in Example 6, compound HQY-10-69 was obtained.

[0286] 1H NMR (400MHz, DMSO) δ7.82(t,J=8.8Hz,3H),7.62(s,1H),7.06(d,J=8.7Hz,1H),6.81(d,J=8.4Hz,2H),3.90(t ,J=5.8Hz,2H),3.01(s,6H),2.84(s,1H),2.06(t,J=6.9Hz,2H),1.92(t,J=5.7Hz,2H),1.68(t,J=7.3Hz,2H).

[0287] MS-ESI: m / z calculated for C 22 H 22 N4OS,Exact Mass:390.15,found 391.01[M+H] + .

[0288] Synthesis of compound HQY-10-71

[0289] By replacing the raw material 1,2-dibromoethane with the photocrosslinking intermediate PC1, and following the other reaction steps as described in Example 6, compound HQY-10-71 was obtained.

[0290] 1 H NMR (400MHz, DMSO) δ7.80(s,1H),7.77(d,J=8.8Hz,2H),7.60(d,J=2.3Hz,1H),7.05(dd,J=8.9,2.4Hz,1H),6.64(d,J=8.7Hz,2H),3 .90(t,J=6.1Hz,2H),2.84(t,J=2.6Hz,1H),2.50(s,3H),2.06(td,J=7.3,2.5Hz,2H),1.92(t,J=6.0Hz,2H),1.68(t,J=7.4Hz,2H).

[0291] MS-ESI: m / z calculated for C 21 H 20 N4OS,Exact Mass:376.14,found 376.99[M+H] + .

[0292] Synthesis of compound HQY-10-90

[0293] By replacing the raw material 1,2-dibromoethane with the photocrosslinking intermediate PC1, and following the other reaction steps as described in Example 6, compound HQY-10-90 was obtained.

[0294] 1 H NMR (400MHz, DMSO) δ7.94(d,J=9.0Hz,2H),7.56(d,J=8.7Hz,1H),7.33(d,J=2.1Hz,1H),6.94(d,J=2.3Hz,1H),6.85(d,J=9.0Hz ,2H),3.90(t,J=6.1Hz,2H),3.03(s,6H),2.84(t,J=2.6Hz,1H),2.09–2.01(m,2H),1.91(t,J=6.0Hz,2H),1.68(t,J=7.4Hz,2H).

[0295] MS-ESI: m / z calculated for C 22 H 22 N4O2, Exact Mass:374.17, found 375.03[M+H] + .

[0296] Synthesis of compound HQY-10-73

[0297] By replacing the raw material 1,2-dibromoethane with the photocrosslinking intermediate PC1, and following the other reaction steps as described in Example 6, compound HQY-10-73 was obtained.

[0298] 1 H NMR (400MHz, DMSO) δ8.15–8.06(m,2H),7.94(d,J=9.1Hz,1H),7.73(d,J=2.7Hz,1H),7.40(t,J=8.7Hz,2H),7.14(dd,J=8.9,2.6 Hz,1H),3.93(t,J=6.1Hz,2H),2.85(t,J=2.6Hz,1H),2.06(td,J=7.5,2.7Hz,2H),1.94(t,J=6.1Hz,2H),1.69(t,J=7.4Hz,2H).

[0299] MS-ESI: m / z calculated for C 20 H 16 FN3OS,Exact Mass:365.10,found 366.1[M+H] + .

[0300] Synthesis of compound HQY-17-15

[0301] By replacing the raw material 1,2-dibromoethane with the photocrosslinking intermediate PC1, and following the other reaction steps as described in Example 6, compound HQY-17-15 was obtained.

[0302] 1 H NMR (400MHz, DMSO) δ8.80 (d, J=2.3Hz, 1H), 8.16 (dd, J=8.8, 2.3Hz, 1H), 7.61 (d, J= 8.9Hz,1H),7.26(d,J=2.5Hz,1H),6.93(dd,J=8.8,2.5Hz,1H),6.52(d,J=8.9Hz,1H ),4.10(t,J=7.5Hz,4H),3.88(t,J=6.1Hz,2H),2.84(t,J=2.7Hz,1H),2.39(p,J=7. 4Hz,2H),2.05(td,J=7.4,2.6Hz,2H),1.90(t,J=6.0Hz,2H),1.68(t,J=7.4Hz,2H).

[0303] MS-ESI: m / z calculated for C 22 H 21 N5O2,Exact Mass:387.17,found 388.1[M+H] + .

[0304] Synthesis of compound HQY-11-62

[0305] By replacing the raw material 1,2-dibromoethane with diethyl bromofluoromethylphosphonate, and following the other reaction steps as described in Example 6, compound HQY-11-62 was obtained.

[0306] 1 H NMR (400MHz, DMSO) δ7.97(d,J=8.9Hz,2H),7.71(d,J=8.6Hz,1H),7.62(d,J=2.0Hz,1H),7.45(s,0.2 5H),7.26(s,0.5H),7.18(dd,J=8.6,2.4Hz,1H),7.08(s,0.25H),6.86(d,J=9.0Hz,2H),3.04(s,6H).

[0307] MS-ESI: m / z calculated for C 16 H 14 F2N2O2,Exact Mass:304.10,found 305.1[M+H] + .

[0308] Synthesis of compound HQY-19-10

[0309] The starting material 1,2-dibromoethane was replaced with 3-bromomethyl-3-methyloxetane, and the other reaction steps were the same as in Example 6, to obtain compound HQY-19-10.

[0310] 1 H NMR (400MHz, DMSO) δ7.94(d,J=8.9Hz,2H),7.57(d,J=8.7Hz,1H),7.39(d,J=2.1Hz,1H),6.98(dd,J=8.7,2.3Hz, 1H), 6.85 (d, J = 9.0Hz, 2H), 4.52 (d, J = 5.7Hz, 2H), 4.33 (d, J = 5.8Hz, 2H), 4.12 (s, 2H), 3.03 (s, 6H), 1.39 (s, 3H).

[0311] MS-ESI: m / z calculated for C 20 H 22 N2O3, Exact Mass:338.16, found 339.2[M+H] + .

[0312] Synthesis of compound HQY-19-11

[0313] The starting material 1,2-dibromoethane was replaced with 3-bromomethyl-3-hydroxymethyl-1-oxecyclobutane, and the other reaction steps were the same as in Example 6, to obtain compound HQY-19-11.

[0314] 1 H NMR (400MHz, DMSO) δ7.94(d,J=8.9Hz,2H),7.56(d,J=8.7Hz,1H),7.39(d,J=2.0Hz,1H),6.97(dd,J= 8.6, 2.2Hz, 1H), 6.85 (d, J = 8.9Hz, 2H), 4.43 (q, J = 5.9Hz, 4H), 4.22 (s, 2H), 3.73 (s, 2H), 3.03 (s, 6H).

[0315] MS-ESI: m / z calculated for C 20 H 22 N2O4, Exact Mass:354.16, found 355.2[M+H] + .

[0316] Synthesis of compound HQY-19-12

[0317] The starting material 1,2-dibromoethane was replaced with 4-bromo-2-methylbut-2-ol, and the other reaction steps were the same as in Example 6, to obtain compound HQY-19-12.

[0318] 1 H NMR (400MHz, DMSO) δ7.93(d,J=8.9Hz,2H),7.54(d,J=8.7Hz,1H),7.33(d,J=2.1Hz,1H),6.92(dd,J=8.6, 2.2Hz, 1H), 6.85 (d, J = 9.0Hz, 2H), 4.14 (t, J = 7.2Hz, 2H), 3.03 (s, 6H), 1.87 (t, J = 7.1Hz, 2H), 1.18 (s, 6H).

[0319] MS-ESI: m / z calculated for C 20 H 24 N2O3, Exact Mass:340.18, found 341.2[M+H] + .

[0320] Synthesis of compound BFT-4-27

[0321] The starting material 1,2-dibromoethane was replaced with 3-bromoepoxide, and the other reaction steps were the same as in Example 6, to obtain compound BFT-4-27.

[0322] 1 H NMR (400MHz, DMSO) δ7.86(d,J=8.9Hz,2H),7.51(d,J=8.6Hz,1H),7.09(d,J=2.2Hz,1H),6.80(dd,J=8.5,2.1Hz, 1H), 6.78 (d, J = 9.0Hz, 2H), 5.27 (dd, J = 10.8, 5.4Hz, 1H), 4.90 (t, J = 6.6Hz, 2H), 4.61–4.44 (m, 2H), 2.96 (s, 6H).

[0323] MS-ESI: m / z calculated for C 18 H 18 N2O3, Exact Mass:310.13, found 311.1[M+H] + .

[0324] Synthesis of compound BFT-4-34

[0325] The starting material 1,2-dibromoethane was replaced with 3-bromo-1,2-propanediol, and the other reaction steps were the same as in Example 6, to obtain compound BFT-4-34.

[0326] 1 H NMR (400MHz, DMSO) δ7.94(d,J=8.9Hz,2H),7.55(d,J=8.6Hz,1H),7.32(d,J=2.0Hz,1H),6.94(dd,J=8.6,2.1Hz,1H),6.85(d,J=8. 9Hz,2H),4.07(dd,J=9.8,3.9Hz,1H),3.96–3.92(m,1H),3.61–3.55(m,1H),3.52–3.46(m,1H),3.83(d,J=5.1Hz,1H),3.03(s,6H).

[0327] MS-ESI: m / z calculated for C 18 H 20 N2O4,Exact Mass:328.14,found 329.1[M+H] + .

[0328] Biological Test Example 1: Effect of Compounds on Phosphorylation of Serine 129 of α-syn, a Marker of Amyloid Aggregation

[0329] Research reports indicate abnormal accumulation of phosphorylation at serine 129 of α-syn in the brains of Parkinson's patients and in transgenic animal models of synucleinosis. This suggests that this post-translational modification may play an important role in the brain, potentially participating in α-syn aggregation, inducing Lewy body formation, and ultimately leading to dopaminergic neuronal degeneration. Western blot analysis of α-syn S129 phosphorylation can provide a preliminary assessment of intracellular α-syn protein aggregation.

[0330] α-syn PFF is a product of ultrasonically broken and purified α-syn fibers in vitro. It has the ability to induce the formation of α-syn aggregates and has been widely used to establish in vivo and in vitro Parkinson's disease models.

[0331] The experimental conditions and procedures for α-syn aggregation in HEK293T cells: HEK293T cells were cultured in vitro and, after reaching the logarithmic growth phase, were digested and collected. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6 / mL. Add 2ml of cells to each well of a 6-well cell culture plate, along with 1μg of pCAGGSα-syn WT-FLAG plasmid and PEI transfection reagent mixture (plasmid:PEI = 1:3m / v). Incubate in a cell culture incubator (37℃, 5% CO2). After 24h, digest and collect the cells, adjusting the cell concentration to 2×10⁶. 5 / mL. Add 1ml of cells to each well of a 12-well cell culture plate, and add α-syn pff in 1X PBS or the same volume of 1X PBS to a final concentration of 10nM. Simultaneously, add compounds (HQY-10-27, HQY-12-16, HQY-8-57, Anle 138b) to a final concentration of 0.5 μM, or compounds HQY-10-27 to final concentrations of 1 μM, 0.5 μM, 0.25 μM, or 0.125 μM, or add compounds HQY-10-27 to a final concentration of 0.5 μM on the second or third day after seeding. After 72 h, wash twice with pre-cooled PBS solution, aspirate the solution, add 500 μL of 1% NP40 cell lysis buffer, protease inhibitor, and phosphatase inhibitor to the wells, transfer to sample tubes, and incubate at 4°C on a shaker for 45 min. Centrifuge at 15000 rpm at 4°C for 45 min to separate the supernatant and precipitate. Wash the precipitate twice with pre-cooled PBS solution, add 1% SDS cell lysis buffer, and lyse. Heat in a metal bath at 95°C for 10 min after lysis. Collect the supernatant and precipitate lysis buffer, use the BCA protein quantification kit to detect the protein content of each group, and use the corresponding lysis buffer to adjust the protein content for Western blot identification.

[0332] Western blot: Add 100 μL of 2X protein loading buffer to 100 μL of cell lysis buffer and heat at 95 °C for 10 min. After the sample cools, perform electrophoresis on an SDS-PAGE (12%) gel at 60 V for 30 min, then switch to 120 V until the leading band reaches the bottom of the gel. Using a turbo semi-dry transfer system, transfer the protein from the gel to a 0.2 μm NC membrane at a constant current of 0.3 A for 120 min. Block the NC membrane with 5% skim milk powder (TBST solution) at room temperature for 1 h, and incubate overnight at 4 °C with phosphorylated S129α-syn antibody. Wash three times with TBST for 10 min each time. Incubate with the appropriate secondary antibody at room temperature for 2 h. Wash three times with TBST for 10 min each time. Incubate with ECL chemiluminescence buffer and detect the luminescence signal.

[0333] The compounds were tested for activity in 293T cells. Their ability to inhibit α-syn aggregate formation was compared by analyzing the minimum effective concentration (c) at which they inhibited S129 phosphorylation by more than 50%. ("+++": c ≤ 0.25 μM, "++": 0.25 μM < c ≤ 0.5 μM, "+": 0.5 μM < c ≤ 1 μM, "-": c ≥ 1 μM)

[0334] Table A. Compound Structure and Activity

[0335] Primary neuron experimental conditions and procedures: SD rats at 16 days of gestation were euthanized by cervical dislocation after isoflurane anesthesia. The fetuses were removed and placed on ice. The left and right hemispheres were dissected and preserved in pre-chilled HBSS buffer. The blood membrane in the brain was removed in liquid, and the brain was washed twice with pre-chilled HBSS buffer. The brain was minced and transferred to 1.5 mL EP tubes. A mixture of papain and nuclease infiltrating the tissue was added, and the tubes were incubated in a cell culture incubator (37℃, 5% CO2) for 30 min. The digested cells were aspirated, filtered through a 40 μM sieve, resuspended in DMEM medium (10% serum), centrifuged at 900 rpm at room temperature for 5 min, and the supernatant was discarded. The cell concentration was adjusted to 4 × 10⁶ cells / mL. 4 / mL, add 1ml of cells to each well of a 12-well cell culture plate coated with poly-L-Lysine, and incubate for 3 hours. Replace the medium with Nerobasal medium (1% GluMax, 1% penicillin / streptomycin, 2% B27). After one week of culture, add α-syn pff to a final concentration of 200nM, and compounds HQY-10-27 and HQY-12-16 to final concentrations of 0.1μM, 0.5μM, and 1μM, respectively.

[0336] Immunofluorescence assay: After 14 days of neuronal drug treatment, cells were washed twice with pre-cooled PBS solution, the solution was aspirated, and 500 μL of 4% PFA was added to each well. The cells were incubated at room temperature for 10 min, followed by three 10-min PBS washes. 0.1% Triton 100 was added to PBS, and the cells were incubated at room temperature for 10 min, followed by three 10-min PBS washes. 5% FBS was added to PBS, and the cells were blocked at room temperature for 1 h, followed by three 10-min PBS washes. Phosphorylated s129α-syn antibody and MAP2 antibody were incubated overnight at 4°C. The cells were washed three times with PBS for 10 min each. Alexa Fluor 488 / 562 fluorescent secondary antibody was incubated at room temperature for 1 h, followed by three 10-min PBS washes. A final concentration of 1 μg / mL DAPI was added to PBS, and the cells were incubated at room temperature for 10 min, followed by three 10-min PBS washes. Cells were fixed onto glass slides using Prolong GOLD mounting medium and air-dried at room temperature in the dark. Imaging was performed using a Leica TCS SP8 confocal microscope system. The fluorescence images were analyzed using ImageJ software to determine the grayscale intensity of phosphorylated S129α-syn and MAP2, and the ratio of phosphorylated S129α-syn to MAP2 was calculated. For three replicate experiments, statistical significance was determined using Graphpad software and one-way ANOVA, with a p-value of **** (p < 0.0001).

[0337] Figure 1 shows the effect of representative compounds on the phosphorylation of serine at position 129 of α-syn. The results indicate that HQY-10-27 and the lead compound HQY-8-57 effectively promoted the reduction of NP40-insoluble α-syn protein and its phosphorylation at a concentration of 0.5 μM. Compared with Anle 138b, a compound reported to inhibit α-syn aggregation, HQY-10-27 had a greater effect on the total amount of α-syn protein and its phosphorylation at the same concentration.

[0338] Figure 2 shows that the effect of compound HQY-10-27 is concentration- and time-dependent. Experimental results indicate that HQY-10-27 has a relatively small effect on α-syn protein and phosphorylation at 0.125 μM, and this effect increases with increasing concentration. After 24 h of treatment with HQY-10-27, the levels of α-syn protein and phosphorylation begin to decrease, and the effect is more pronounced at 72 h.

[0339] Figure 3 shows the effects of different concentrations of compounds on the formation of fibrillary aggregates in neurons induced by α-syn pff. Compared with the PBS-treated group, α-syn pff stimulation of neurons significantly increased phosphorylation of S129α-syn, and compound HQY-10-27 significantly inhibited phosphorylation of S129α-syn at 0.5 μM and 1 μM, while MAP2 protein was unaffected. The negative control compound HQY-12-16 had almost no effect on phosphorylation of S129α-syn.

[0340] Figure 4 shows the statistical analysis of the fluorescence experiment.

[0341] Biological Test Example 2: Verification of Protein Binding to Compound HQY-10-27

[0342] To investigate how compound HQY-10-27 eliminates asyn amyloid protein aggregation in vivo, we synthesized a probe molecule, HQY-10-71, with a photocrosslinking alkyne group. The carbene is generated by the diazirine group under 365 nm UV irradiation. The carbene inserts into an alcohol hydroxyl or amino group, thereby coupling to a protein that interacts with a small molecule. Further biotin labeling of the compound was achieved via copper-catalyzed biotin-azide and alkyne cycloaddition. After affinity purification, we obtained the small target protein through proteomic analysis.

[0343] Traditional competitive pulldown experiments and surface plasmon resonance techniques helped determine the proteins that the small molecule HQY-10-27 can bind to and the affinity constants between them.

[0344] In vitro photocrosslinking-click chemistry-pulldown experimental conditions and procedures: HEK293T cell culture and pCAGGSα-syn-WT-FLAG plasmid transfection were performed as described above. After 24 hours, cells were digested and collected, and the cell concentration was adjusted to 2×10⁻⁶. 5 / mL. Add 10 mL of cells to each well in a 10 cm cell culture dish, and add α-syn pff in 1X PBS to a final concentration of 10 nM. On the third day after seeding, add compound HQY-10-71 or the same volume of DMSO to a final concentration of 0.25 μM. After 24 h, wash twice with pre-cooled PBS solution, and add 1 mL of 0.25 μM compound HQY-10-71 / DMSO in PBS. Place on ice and ligate in UV for 30 min, wash twice with pre-cooled PBS solution, add 1 mL of 1% SDS cell lysis buffer to each dish, and lyse. Heat in a metal bath at 95 °C for 10 min. Prepare the catalyst for the click chemistry reaction (100 μM Biotin-N3, 100 μM TBTA, 1 mM CuSO4 / CuBr, 1 mM TCEP), mix well, and add to the reaction solution. Incubate at 25 °C for 1 h. After the reaction was terminated, add 4 volumes of methanol, 1 volume of chloroform, and 3 volumes of double-distilled water, vortex to mix, and incubate for 40 min. Then centrifuge at 15,000 rpm for 15 min at 4°C. Wash twice with pre-cooled methanol, incubate at 4°C for 25 min to remove methanol, dry, and redissolve in 1% SDS cell lysis buffer. Analyze the protein content of each group using a BCA protein quantification kit, and adjust the protein levels using the corresponding lysis buffer. Dilute the lysis buffer with Tris buffer (50 mM Tris, 150 mM NaCl, pH 7.4) to a final SDS concentration of 0.1%. Add 5 μL of streptavidin-coated beads to each lysis buffer tube, mix at 4°C, and incubate overnight. Centrifuge to remove the supernatant, and wash the affinity beads five times with buffer (0.1% SDS, 50 mM Tris, 150 mM NaCl, pH 7.4) for 10 min each time. Remove as much supernatant as possible and store at -20°C.

[0345] Sample preparation and LC-MS / MS detection: Add an appropriate amount of 8M Urea in 100mM Tris to the beads, pH 8.5, shake and incubate, then add 100mM TCEP to a final concentration of 5mM, and incubate at 37°C for 20 min. Add 500mM IAA to a final concentration of 10mM, and incubate at 37°C for 20 min in the dark. Dilute the 8M Urea to a final concentration of 2M, add calcium chloride to a final concentration of 1mM and trypsin at a 1:100 ratio, and incubate overnight at 37°C with shaking. Add 90% formic acid to terminate the enzymatic digestion, centrifuge at 15000 rpm at room temperature for 15 min. Transfer the supernatant to a C18 column, centrifuge at 2000g, wash with an appropriate amount of 0.1% FA, elute with 100 μL of elution buffer (70% ACN + 0.1% FA in 100mM Tris, pH 8.5) and collect. After repeating the process three times, the collected elution solution was evaporated for 2 hours.

[0346] Samples were analyzed using an online EASY-nLC 1000 HPLC coupled with a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Mixtures were directly loaded onto a 15 cm self-made capillary column (100 μm inner diameter, C18-AQ 1.9 μm resin) and separated by gradient at a flow rate of 300 nL / min. Mobile phase A consisted of 0.1% FA, 2% acetonitrile, and 98% H₂O, while mobile phase B consisted of 0.1% FA, 2% H₂O, and 98% acetonitrile. Analysis was performed in data-dependent mode by a single full scan (m / z: 350–1500; resolution: 15,000; AGC target: 3,000,000 and maximum injection time: 20 ms), followed by a two-wave MS scan (32% normalized collision energy; AGC target: 100,000; maximum injection time: dynamic).

[0347] MS / MS raw spectra were processed using MaxQuant software (version 1.6.0.1). The SwissProt human protein sequence database was used for database searching. Trypsin was set as the enzyme, and the maximum missed cleavage was set to 2. The false discovery rate at both the peptide matching level and the protein level was controlled to be below 1%. The mean protein intensity for each treatment group was calculated using Graphpad Prism 8.0.2, and the change in concentration of compound HQY-10-71 compared to the solvent group was obtained. Log2 and -Log10 of the fold change (FC) and p-values ​​were plotted. All tests were two-tailed t-tests assuming equal variances.

[0348] Surface Plasmon Resonance (SPR): SPR data were collected on a Biacore 8K device (GE Healthcare #29215379). Immobilized on a CM5 biosensor chip (GE Healthcare) with 10 mM acetate solution (GE Healthcare), pH 4.5, the final surface ligand density was approximately 8000–15000 response units (RU). Briefly, the entire immobilization process used a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), protein sample, and ethanolamine solution, injected into the flow cells of the Biacore chip at a flow rate of 10 μL / min. The carboxyl groups on the sensor chip surface were activated by injecting 200 μL of a 1:1 (v / v) EDC / NHS mixture (included in the amine coupling kit, Cytiva Life Sciences) into flow cells 1 and 2, followed by injecting protein into overflow cell 2 for 200 seconds. Residual activating groups in both flow cells were blocked by injecting 129 μL of 1M ethanolamine-HCl, pH 8.5. The antibody-coated sensor chip was equilibrated with PBS buffer before starting the binding assay. At 25°C, serially diluted solutions of compounds ranging from 20 μM to 0.625 μM in PBS buffer were injected into both flow cells at a flow rate of 30 μL / min. The contact time (binding phase) for each sample cycle was 120 seconds, followed by a dissociation time of 600 seconds. After each injection cycle, the Biacore chip was rinsed with PBS buffer before starting the next cycle.

[0349] Figure 5 shows the volcano plot of the proteomic results of compound HQY-10-71. The target protein α-syn, as well as HSP70, HSP40 and other HSP family proteins, are highlighted.

[0350] Figure 6 shows the results of the competitive pulldown experiment. After treatment of cells with compound HQY-10-27, the binding ability of probe HQY-10-71 to α-syn and HSP70 / HSP40 proteins was weakened, and comparable to that of the control compound HQY-10-73. This indicates that HQY-10-27 can bind to the target proteins intracellularly. After treatment with the control compound HQY-12-16, the protein binding ability of probe HQY-10-71 remained unchanged.

[0351] Figures 7a-7d and Table 1 show the kinetic curves of the compounds and proteins, as well as their binding constants.

[0352] Table 1

[0353] Example of biological testing 3: Compound HQY-10-27 promotes the binding of HSP40 protein to α-syn pff and inhibits fiber growth.

[0354] HSP40 has been reported to aid HSP70 in clearing misfolded or polyproteins. Enzyme-linked immunosorbent assays (ELISA) can be used to test which protein-protein interactions a compound promotes. Furthermore, α-synuclein filament growth can be labeled with the fluorescent dye Thioflavin T, ultimately revealing how compounds affect protein aggregation over time.

[0355] Enzyme-linked immunosorbent assay (ELISA): High-binding 96-well plates (Corning Costar, catalog number: 3590) were coated overnight at 4°C with 100 μL of HSP40 protein PBS solution. After blocking with 5% milk PBS solution and 0.05% Tween-20, 100 μL of α-syn pff and compounds HQY-10-27 / HQY-12-16 were added to a final concentration of 100 μM and 10 μM, respectively. The reaction was then terminated with 2M sulfuric acid after color development using anti-α-syn primary antibody (Abcam, ab138501, 1:2000 dilution) and horseradish peroxidase-conjugated secondary antibody (Abcam, ab31460, 1:10000 dilution) and TMB substrate reagent kit (BD OptEIA, 555214). Statistical significance was determined using Graphpad software and one-way ANOVA, p < 0.0001.

[0356] Thioflavin T assay: 100 μM α-syn protein and 1 μM HSP40 protein were incubated in 25 mM Na2HPO4 / NaH2PO4, pH 6.5, 0.05% w / v NaN3, 50 μM ThT, and 0.5 μM α-syn PFF. All experiments were performed in NUNC 384 plates and stirred at 37°C with constant stirring at 900 rpm (dual-track). ThT fluorescence signals were measured using a microplate reader (BMG Labtech), with excitation at 440 nm and emission at 480 nm. Each experiment consisted of three biological replicates, with a minimum of three replicates. Statistical significance was determined using Graphpad software and one-way ANOVA, with ****, p < 0.0001.

[0357] Figure 8 shows that the compound can promote the binding of HSP40 protein to α-syn pff. At 100 μM, the protein-protein interaction was significantly enhanced by the compound HQY-10-27, while the control compound HQY-12-16 at the same concentration had no effect.

[0358] Figures 9a and 9b show that compound HQY-10-27 can inhibit α-synuclein fiber growth. When the ratio of α-synuclein protein to HSP40 protein was 100:1, the addition of 10 μM, 30 μM, and 100 μM of compound HQY-10-27 significantly inhibited fiber proliferation. Meanwhile, the control compound HQY-12-16 showed no significant effect.

[0359] Biological Test Example 4: The Role of Compounds in an α-syn pff-induced Parkinson's Disease Mouse Model

[0360] Mice are the most commonly used model organism in Parkinson's disease research. One research group reported that injecting in vitro purified α-syn protein pff into the mouse brain could induce Parkinson's disease symptoms in mice within several months, resulting in pathological α-syn protein aggregation and significantly increased phosphorylated S129α-syn in brain regions such as the substantia nigra and motor nuclei. Therefore, we established this animal model to study the efficacy of compound HQY-10-27 in vivo.

[0361] Establishment of a Parkinson's Disease Mouse Model: Six-week-old C57BL / 6J mice were prepared and housed individually. Three days before the scheduled surgery date, mice were trained to eat peanut butter, and those that could eat independently were observed and selected. On the day of surgery, after anesthesia, the mice's brains were prepared, and a single needle was inserted into the right forebrain (coordinates: +0.2 mm relative to the anterior fontanelle, +2.0 mm from the midline) to inject 4 μg α-syn pff (2.5 μL) unilaterally into the dorsal striatum (2.6 mm below the dura mater). The injection was performed using a 10 μL syringe (Hamilton, NV) at a rate of 0.4 μL / min. Three days post-surgery, mice resumed a normal diet. Body weight was measured and recorded two to three times per week. Compound HQY-10-27 powder was mixed with peanut butter at a dose of 5 mpk and administered daily at fixed times in the morning, with mice's self-administration recorded. The control group received only the same amount of peanut butter. Oral administration was discontinued in the fourth month after model establishment.

[0362] Grasp Force Test: Grasp force tests were conducted on mice six months after model establishment. The experimental mice were placed on a gripper grid, with their trunks horizontal, and only their forepaws were allowed to remain attached to the grid before any measurements were taken. The mouse's tail was gently pulled back, ensuring the mouse gripped the top of the grid and its trunk remained horizontal, and the maximum grip force value displayed on the screen was recorded. This process was repeated twice to obtain three forepaw grip force measurements. The maximum grip force was recorded before release. The average performance of the animals in each trial is expressed as the average of the three trials. Statistical significance was determined using Graphpad software and one-way ANOVA, ****, p < 0.0001.

[0363] Immunohistochemistry:

[0364] At the end of the six-month model establishment period, brains were harvested from mice. The entire brain was perfused with PBS using a peristaltic pump and then fixed in 4% PFA. After dehydration using an automated dehydrator, the brain tissue was embedded in paraffin blocks, cut into 6 μm sections, mounted on glass slides, and dried at 60°C for storage. The slides were washed twice consecutively in xylene for 5 min each time, followed by washing sequentially in 100%, 100%, 95%, 80%, and 70% ethanol for 1 min each to dewax. The slides were then incubated in deionized water for 1 min, immersed in citric acid (pH 6.0), and heat-retarded for 15 min. Finally, the slides were incubated in 5% hydrogen peroxide (methanol) in methanol to inhibit endogenous peroxidase activity. The slides were washed in running tap water for 10 min, washed in 0.1 M Tris for 5 min, and then blocked in 0.1 M Tris / 2% fetal bovine serum (FBS). Slides were incubated overnight with phosphorylated S129α-syn antibody. The slides were washed with 0.1M Tris for 5 min with the primary antibody, then incubated with goat anti-rabbit biotinylated IgG for 1 h. The slides were washed with 0.1M Tris for 5 min to remove the biotinylated antibody, then incubated with avidin-biotin solution (Vector PK-6100) for 1 h. The slides were then washed with 0.1M Tris for 5 min, developed with ImmPACT DAB peroxidase substrate (Vector SK-4105), and briefly counterstained with hematoxylin. The slides were rinsed with running tap water for 5 min, dehydrated with 70%, 80%, 95%, 100%, and 100% ethanol for 1 min each, then rinsed twice in xylene for 5 min each time, and mounted with resin mounting medium. Brain slices were recorded using a 3D HISTECH pathology scanner with a 20x microscope. Pathological images were analyzed using imajej software to obtain the ratio of phosphorylated S129α-syn area to brain region area. Graphpad software was used for one-way ANOVA to statistically analyze the differences; p < 0.0001.

[0365] Figure 10 shows the weight changes in a Parkinson's disease model mouse. Compound HQY-10-27 had little effect on mouse weight after four months of administration, and no significant long-term side effects were observed after discontinuation of the drug.

[0366] Figure 11 shows the changes in grip strength in mice. In the grip strength test at six months, the mice that were orally administered the drug showed significantly stronger grip strength, and their limb grip strength approached that of the non-disease control mice.

[0367] Figures 12 and 13 show pathological phosphorylation of S129α-syn in mouse brain regions. Oral administration of HQY-10-27 significantly suppressed the area of ​​phosphorylated S129α-syn in the substantia nigra and motor cortex, indicating that the possible accumulation of pathological proteins in the mouse brain was controlled. Consequently, the behavior of the mice was also improved.

[0368] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound of Formula I, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, wherein X1 is either O or S; X2 and X3 are independently CH or N; R1is C1-C8alkoxy, C1-C8haloalkoxy, A 4-6 membered heterocyclic group consisting of a C1-C8 alkoxy group substituted with 1, 2, or 3 hydroxyl groups, or a -O-C0-C2 alkylene group containing a heteroatom selected from O, N, and S, wherein the heterocyclic group is optionally substituted with 1, 2, or 3 groups selected from the group consisting of OH, C1-C3 alkyl, and -C1-C3 alkylene-OH; R2 is H, halogen, C1-C4 alkyl, or C1-C4 haloalkyl; R3 is H, C1-C4 alkyl, or C1-C4 haloalkyl; R4 is a C1-C4 alkyl or a C1-C4 haloalkyl; Alternatively, R3 and R4 together with the N atoms attached to them can form a 3-6 membered heterocyclic group containing one N heteroatom.

2. The compound of claim 1, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, wherein X2 is CH, and X3 is CH.

3. The compound of claim 1, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, wherein X1 is 0.

4. The compound of claim 1, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, wherein R1is C1-C4alkoxy, C1-C4haloalkoxy, 1 or 2 hydroxy-substituted C1-C6alkoxy, -O-4- to 5-membered heterocyclyl containing 1 O heteroatom, -O-C1-C2alkylene-4- to 5-membered heterocyclyl containing 1 O heteroatom, wherein said heterocyclyl is independently optionally substituted with 1 or 2 groups selected from the group consisting of OH, C1-C2alkyl, and -C1-C2alkylene-OH.

5. The compound of claim 1, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, wherein R1is C1-C4alkoxy, C1-C4haloalkoxy or 6. The compound of claim 1, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, wherein R1is selected from the group consisting of methoxy, difluoromethoxy, trifluoromethoxy, 7. The compound of claim 1, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, wherein R2 is H, a halogen, or a halomethyl group, preferably H, F, Cl, Br, or a trifluoromethyl group.

8. The compound of claim 1, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, wherein R3 is H, C1-C4 alkyl and R4 is C1-C4 alkyl, or R3 and R4 together with the N attached to them form a 3-6 membered heterocyclic group containing one N heteroatom.

9. The compound of claim 1, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, wherein when X1is S, R3and R4together with the N to which they are attached form a 3-6 membered heterocyclyl containing one N heteroatom or R1is 10. The compound of claim 1, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, wherein When X1 is 0, R2 and R3 are not simultaneously H.

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

12. A pharmaceutical composition comprising: a. The compound of claim 1, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof; and b. A pharmaceutically acceptable carrier.

13. Use of the compound of claim 1, its pharmaceutically acceptable salt, or a deuterated compound thereof, or the pharmaceutical composition of claim 8 in the preparation of an α-synuclein aggregate inhibitor.

14. Use of the compound of claim 1, a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or the pharmaceutical composition of claim 8, in the preparation of a medicament for the prevention or treatment of neurodegenerative diseases associated with α-synuclein aggregates or misfolded protein aggregates thereof.

15. The use according to claim 14, characterized in that, Neurodegenerative diseases are selected from the following group: Parkinson's disease, multiple system atrophy, Lewy body dementia, Alzheimer's disease, or amyotrophic lateral sclerosis.

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