CN117384022A

CN117384022A - Deuterated compounds and uses thereof

Info

Publication number: CN117384022A
Application number: CN202310849865.4A
Authority: CN
Inventors: 张学军; 胡文兵; 钱丽娜; 杨洋; 卓君明; 叶大炳; 刘礼飞; 李莉娥; 杨俊�
Original assignee: Humanwell Healthcare Group Co ltd; Wuhan Humanwell Innovative Drug Research and Development Center Ltd Co
Current assignee: Humanwell Healthcare Group Co ltd; Wuhan Humanwell Innovative Drug Research and Development Center Ltd Co
Priority date: 2022-07-11
Filing date: 2023-07-11
Publication date: 2024-01-12
Also published as: WO2024012440A1

Abstract

The invention discloses a deuterated compound and application thereof. The invention specifically provides a compound shown in a formula I, a tautomer, a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof. The compounds of the invention are useful for the aggregation of abnormal proteins such as mSOD1 proteinThe nerve cell injury induced by the collection has a protective effect and excellent pharmacokinetic property; the compound has good effect of preventing and/or treating ALS.

Description

Deuterated compounds and uses thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a deuterated compound and application thereof.

Background

Amyotrophic lateral sclerosis (ALS, also known as "freezing syndrome") is a rare neurodegenerative disease, which is caused by degeneration of Upper Motor Neurons (UMN), and is also an irreversible lethal motor neuron disease, and no radical method exists at present, and has been listed as one of five absolute symptoms worldwide by the world health organization. The research results show that abnormal folding and accumulation of protein can be an important cause of amyotrophic lateral sclerosis, wherein the mSOD1 toxicity generated by misfolding of mutant SOD1 protein can cause motor neuron degeneration and further progress to ALS; in addition, abnormal accumulation of mutant TDP-43 protein was observed in nerve bundles of sporadic ALS patients, and nerve cells were considered to be toxic, which is one of the causes of ALS pathogenesis. On an animal model, the abnormal aggregation of mSOD1 and TDP-43 proteins can be inhibited by medicaments to play a role in neuroprotection, reduce UMN injury caused by the mSOD1 and the TDP-43 proteins, stop ALS process (Baris et al, clin Transl Med,2021, 11:e336), have a remarkable improvement effect on the ALS animal model, and provide a new treatment direction for the treatment of ALS. Thus, the development of new neuroprotective compounds has positive implications for the treatment of disease.

Disclosure of Invention

The invention aims to solve the technical problem that few existing medicines for treating diseases related to abnormal protein aggregates are overcome, and provides a deuterated compound and application thereof. The deuterated compound has a protective effect on nerve cell injury induced by abnormal protein aggregation and has excellent pharmacokinetic properties; has good effect in preventing and/or treating ALS.

In a first aspect of the invention, a compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof:

wherein L is selected from unsubstituted or substituted by at least one R ¹ Substituted C _1-6 Alkylene or C _1-6 A halogenated alkylene group; the R is ¹ Selected from hydrogen, deuterium, C _1-6 Deuterated alkyl;

R ^a and R is ^b Each independently selected from hydrogen, deuterium, halogen, hydroxy, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 Alkyl, C _3-8 Cycloalkyl, C _1-6 Deuterated alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, C _3-8 Halogenated cycloalkyl, C _1-6 Alkyl hydroxy, C _1-6 Alkylcarbonyl, C _1-6 Alkoxy, C _1-6 Haloalkoxy, 3-10 membered heterocycloalkyl; the C is _1-6 Alkyl, C _3-8 Cycloalkyl, C _1-6 Deuterated alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, C _3-8 Halogenated cycloalkyl, C _1-6 Alkyl hydroxy, C _1-6 Alkylcarbonyl, C _1-6 Alkoxy, C _1-6 Haloalkoxy, 3-to 10-membered heterocycloalkyl optionally substituted with one or more R ^c Substitution;

the R is ^c A substituent selected from the group consisting of: halogen, hydroxy, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 Alkyl, C _1-6 Deuterated alkyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, C _1-6 Alkyl hydroxy, C _1-6 Alkylcarbonyl, C _1-6 Alkoxy, C _1-6 Haloalkoxy groups; when the substituents are plural, the R ^c The same or different;

m is 0, 1, 2, 3 or 4;

n is 1, 2, 3, 4 or 5; and, in addition, the processing unit,

when L is selected from unsubstituted C _1-6 Alkyl or C _1-6 When haloalkyl, the R ^a And R is ^b At least one of them is selected from deuterium or C _1-6 Deuterated alkyl.

In a second aspect of the invention, a compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof:

wherein L is selected from unsubstituted or substituted by at least one R ¹ Substituted C _1-6 Alkyl or C _1-6 A haloalkyl group; the R is ¹ Selected from hydrogen, deuterium, C _1-6 Deuterated alkyl;

R ^a and R is ^b Each independently selected from hydrogen, deuterium, halogen, hydroxy, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 Alkyl, C _3-8 Cycloalkyl, C _1-6 Deuterated alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, C _3-8 Halogenated cycloalkyl, C _1-6 Alkyl hydroxy, C _1-6 Alkylcarbonyl, C _1-6 Alkoxy, C _1-6 Haloalkoxy, 3-10 membered heterocycloalkyl; the C is _1-6 Alkyl, C _3-8 Cycloalkyl, C _1-6 Deuterated alkyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, C _3-8 Halogenated cycloalkyl, C _1-6 Alkyl hydroxy, C _1-6 Alkylcarbonyl, C _1-6 Alkoxy, C _1-6 Haloalkoxy groups are optionally substituted with one or more R ^c Substitution;

m is 0, 1, 2, 3 or 4;

n is 1, 2, 3, 4 or 5; and, in addition, the processing unit,

In the present invention, certain substituents in the compounds of formula I may be defined as follows, and the substituents not mentioned are defined in any of the schemes above.

As will be appreciated by those skilled in the art, in accordance with the conventions used in the art, in the structural formulae of the present application, For depicting chemical bonds, which are points where a moiety or substituent is attached to a core structure or a backbone structure.

In a preferred embodiment of the invention, the 3-10 membered heterocycloalkyl is monocyclic, fused bicyclic, bicyclic including bridged or spiro bicyclic.

In a preferred embodiment of the present invention, the 3-10 membered heterocycloalkyl further has 1 to 3 heteroatoms selected from N, O, S.

In a preferred embodiment of the present invention, the R ¹ Selected from deuterium and C _1-6 Deuterated alkyl.

In a preferred embodiment of the present invention, the compound of formula I is selected from the following structures:

wherein L is selected from unsubstituted, or substituted by 1 or 2R ¹ Substituted C _1-6 Alkyl or C _1-6 A haloalkyl group; the R is ¹ Selected from deuterium, C _1-6 Deuterated alkyl; r is R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ Each independently selected from hydrogen, deuterium, halogen, hydroxy, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 Alkyl, C _3-8 Cycloalkyl, C _1-6 Deuterated alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, C _3-8 Halogenated cycloalkyl, C _1-6 Alkyl hydroxy, C _1-6 Alkylcarbonyl, C _1-6 Alkoxy, C _1-6 Haloalkoxy, 3-10 membered heterocycloalkyl; the C is _1-6 Alkyl, C _3-8 Cycloalkyl, C _1-6 Deuterated alkyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, C _3-8 Halogenated cycloalkyl, C _1-6 Alkyl hydroxy, C _1-6 Alkylcarbonyl, C _1-6 Alkoxy, C _1-6 Haloalkoxy groups are optionally substituted with one or more R ^c Substitution; the R is ^c A substituent selected from the group consisting of: halogen, hydroxy, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 Alkyl, C _1-6 Deuterated alkyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, C _1-6 Alkyl hydroxy, C _1-6 Alkylcarbonyl, C _1-6 Alkoxy, C _1-6 Haloalkoxy groups; when the substituents are plural, the R ^c The same or different; and when deuterium substitution is not present on L, the R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ At least one of them is selected from deuterium or C _1-6 Deuterated alkyl.

In a preferred embodiment of the invention, said L is substituted with 1 or 2R ¹ Substituted C _1-6 Alkyl, R ¹ Selected from deuterium or C _1-6 Deuterated alkyl; preferably, said R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ H.

In a preferred embodiment of the invention, the L is unsubstituted C _1-6 Alkyl, said R ⁷ Deuterium, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ And R is ⁸ H.

wherein R is ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ Having the definition as described in the compounds of formula II of the present invention; r is R ^1a 、R ^1b Each independently selected from hydrogen, deuterium, C _1-6 Alkyl or C _1-6 Deuterated alkyl; and R is ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ 、R ⁸ 、R ^1a And R is ^1b At least one of which contains deuterium.

In a preferred embodiment of the present invention, the R ^1a 、R ^1b Each independently selected from hydrogen, deuterium, methyl or deuterated methyl.

In a preferred embodiment of the invention, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ 、R ⁸ 、R ^1a And R is ^1b At least one of them is deuterium, or deuterated methyl.

In a preferred embodiment of the invention, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b Each independently is hydrogen or deuterium, and R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b At least one of them is deuterium; r is R ⁶ 、R ⁷ 、R ⁸ 、R ^1b Is hydrogen; r is R ^1a Is methyl.

In a preferred embodiment of the invention, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b Is hydrogen; r is R ^1b Is hydrogen; r is R ^1a Is methyl; r is R ⁶ 、R ⁷ 、R ⁸ Each independently is hydrogen or deuterium, and R ⁶ 、R ⁷ 、R ⁸ At least one of them is deuterium.

In a preferred embodiment of the invention, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ 、R ⁸ Is hydrogen; r is R ^1a 、R ^1b Each independently selected from hydrogen, deuterium, methyl or deuterated methyl, and R ^1a 、R ^1b At least one of which is deuterium or deuterated methyl.

In a preferred embodiment of the present invention, in the compound of formula II, R is ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ Each independently selected from hydrogen, deuterium, halogen, C _1-6 Alkyl, C _1-6 Deuterated alkyl or C _1-6 A haloalkyl group.

In a preferred embodiment of the present invention, the R ^1a And R is ^1b Each independently selected from hydrogen or C _1-6 Alkyl, said R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ Each independently selected from hydrogen or deuterium, and R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ At least one of which is selected from deuterium; preferably, R ⁷ Deuterium, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ And R is ⁸ H.

In a preferred embodiment of the invention, the C _1-6 Deuterated alkyl is deuterated methyl, deuterated ethyl, deuterated propyl, deuterated butyl, deuterated isobutyl, for example, deuterated methyl or deuterated ethyl.

In a preferred embodiment of the invention, the C _1-6 Deuterated alkyl is deuterated methyl.

In a preferred embodiment of the invention, the C _1-6 Alkyl is methyl.

In a preferred embodiment of the invention, the L is selected from the group consisting of-CH (CH) ₃ )-、-CD(CH ₃ )-、-CH(CD ₃ ) -or-CD (CD) ₃ )-。

In a preferred embodiment of the present invention, the R ^a And R is ^b Each independently selected from deuterium, deuterated methyl, or trifluoromethyl.

In a preferred embodiment of the present invention, the compound of formula I is selected from any one of the following compounds:

in a third aspect of the present invention, there is provided a pharmaceutical composition comprising: a compound of formula I as described in the first aspect of the invention, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof; and a pharmaceutically acceptable carrier.

In a fourth aspect of the present invention, there is provided a compound of formula I, a tautomer thereof, a pharmaceutical composition comprising the compound of formula I,

Use of a stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, or use of a pharmaceutical composition according to the third aspect of the invention, the use comprising: preventing and/or treating diseases associated with the presence of protein folding, misfolding, or abnormal protein aggregates; and/or preventing and/or treating neurodegenerative diseases; and/or preventing and/or treating a disease associated with degeneration of upper motor neurons; and/or preparing a medicament, pharmaceutical composition or formulation for the presence of protein folding, misfolding, or abnormal protein aggregate related diseases; and/or preparing a medicament, pharmaceutical composition or formulation for the neurodegenerative disease; and/or preparing a medicament, a pharmaceutical composition or a preparation for preventing and/or treating diseases related to the degeneration of upper motor neurons.

Preferably, the abnormal protein aggregates comprise SOD1 protein aggregates or TDP-43 protein aggregates.

Preferably, the SOD1 protein aggregate is a G93A SOD1 protein aggregate or a G85R SOD1 protein aggregate.

Preferably, the neurodegenerative disease comprises: parkinson's Disease (PD), diffuse Lewy Body Disease (DLBD), multiple System Atrophy (MSA), and pantothenate kinase-associated neurodegeneration (PANK), huntington's Disease (HD), a viral disease (e.g., jacob's disease), alzheimer's Disease (AD), or frontotemporal lobar degeneration (FTLD).

Preferably, the disease associated with degeneration of upper motor neurons includes: amyotrophic Lateral Sclerosis (ALS), primary Lateral Sclerosis (PLS), hereditary Spastic Paraplegia (HSP).

In a fifth aspect of the present invention, there is provided a method for preventing and/or treating a disease associated with the presence of protein folding/misfolding, or abnormal protein aggregates, or preventing and/or treating a neurodegenerative disease, or preventing and/or treating a disease associated with degeneration of upper motor neurons, comprising the steps of: administering to a subject in need thereof a compound of formula I according to the first aspect of the invention, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof, or a pharmaceutical composition according to the second aspect of the invention.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Terminology and definitions

Unless otherwise indicated, the radical and term definitions recited in the specification and claims of this application, including as examples, exemplary definitions, preferred definitions, definitions recited in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. Such combinations and combined group definitions and structures of compounds should fall within the scope of the description herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety unless otherwise indicated. If there are multiple definitions of terms herein, the definitions of this chapter shall control.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the inventive subject matter. In this application, the singular is used to include the plural unless specifically stated otherwise. It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the use of "or" means "and/or" unless stated otherwise. Furthermore, the terms "include," as well as other forms, such as "comprising," "including," and "containing," are not limiting.

Conventional methods within the skill of the art, such as mass spectrometry, NMR, IR and UV/VIS spectroscopy, and pharmacological methods are employed unless otherwise indicated. Unless specifically defined otherwise, the terms used herein in the description of analytical chemistry, organic synthetic chemistry, and pharmaceutical chemistry are known in the art. Standard techniques may be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients. For example, the reaction and purification can be carried out using the manufacturer's instructions for the kit, or in a manner well known in the art or in accordance with the teachings of the present invention. The techniques and methods described above may generally be practiced according to conventional methods well known in the art, based on a number of general and more specific descriptions in the literature cited and discussed in this specification. In this specification, groups and substituents thereof can be selected by one skilled in the art to provide stable moieties and compounds.

When substituents are described by conventional formulas written from left to right, the substituents also include chemically equivalent substituents obtained when writing formulas from right to left. For example, CH ₂ Equivalent of OIn OCH (OCH) ₂ . As used herein,or->Representing the attachment site of the group. As used herein, "R ₁ "," R1 "and" R ¹ "has the same meaning and can be replaced with each other. For R ₂ And the like, and the meanings of like definitions are the same.

The section headings used herein are for purposes of organizing articles only and should not be construed as limiting the subject matter. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, operating manuals, and treatises, are hereby incorporated by reference in their entirety.

In addition to the foregoing, when used in the specification and claims of this application, the following terms have the meanings indicated below, unless specifically indicated otherwise.

Where a range of values recited in the specification and claims is understood to be an "integer," it is understood that both endpoints of the range and each integer within the range are recited. For example, an "integer of 1 to 6" should be understood to describe each integer of 0, 1, 2, 3, 4, 5, and 6. When a numerical range is understood as a "number," it is understood that both endpoints of the range are noted, as well as each integer within the range, and each fraction within the range. For example, a "number of 1 to 10" should be understood to describe not only each integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, but also at least the sum of each integer with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, respectively.

In the present application, "saturated, partially saturated or unsaturated" includes substituents that are saturated, substituents that are fully unsaturated with hydrogen, and substituents that are partially saturated with hydrogen.

In the present application, the term "halogen" means fluorine, chlorine, bromine, iodine, alone or as part of other substituents; fluorine or chlorine is preferred.

As used herein, the term "cyano" means —cn, alone or as part of another substituent.

As used herein, the term "amino" means-NH, alone or as part of another substituent ₂ 。

In this application, the term "hydroxy" means-OH, alone or as part of another substituent.

The term "alkyl" when used alone or as part of another substituent means a straight or branched hydrocarbon chain group consisting of only carbon and hydrogen atoms, having, for example, 1 to 6 carbon atoms, and being attached to the remainder of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl and hexyl. The alkyl group may be unsubstituted or substituted with one or more suitable substituents.

The term "alkylene" when used alone or as part of another substituent is understood to mean a straight or branched chain saturated, unsaturated or partially saturated divalent hydrocarbon radical. For example "C _1-6 Alkylene "or" C _1- C ₆ Alkylene "means a straight or branched chain divalent hydrocarbon radical having 1 to 6 carbon atoms including, but not limited to, methylene, ethylene, propylene, 1-methylpropylene, butylene.

The term "C", alone or as part of another substituent _α-β Haloalkyl "refers to an alkyl group as described above wherein any number (at least one) of the hydrogen atoms attached to the alkyl chain are replaced with fluorine, chlorine, bromine or iodine.

The term "cycloalkyl" alone or as part of another substituent means a cyclic alkyl group. The term "m-n membered cycloalkyl" or "C _m-n Cycloalkyl "is understood to mean a saturated, unsaturated or partially saturated carbocyclic ring having m to n atoms. For example, "3-15 membered cycloalkyl" or "C ₃ -C ₁₅ Cycloalkyl "means a cyclic ring containing 3 to 15,3 to 9,3 to 6 or 3 to 5 carbon atomsAlkyl, which may contain 1 to 4 rings. "3-to 10-membered cycloalkyl" contains 3 to 10 carbon atoms. Including monocyclic, bicyclic, tricyclic, spiro, or bridged rings. Examples of unsubstituted cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl, or a bicyclic hydrocarbon group such as a decalin ring. Cycloalkyl groups may be substituted with one or more substituents. In some embodiments, cycloalkyl groups may be cycloalkyl groups fused to aryl or heteroaryl ring groups. The term "cycloalkyl" may be used interchangeably with the term "carbocyclyl".

The term "heterocycloalkyl" when used alone or as part of another substituent refers to cycloalkyl groups in which one or more (in some embodiments 1 to 3) carbon atoms are replaced with heteroatoms such as, but not limited to N, O, S and P. The term "m-n membered heterocycloalkyl" is understood to mean a saturated, unsaturated or partially saturated ring having m to n atoms, wherein the heterocyclic atoms are selected from N, O, S, P, preferably from N, O or S. For example, the term "4-8 membered heterocycloalkyl" or "C ₄ -C ₈ Heterocycloalkyl "is understood to mean a saturated, unsaturated or partially saturated ring having from 4 to 8 atoms, wherein 1, 2, 3 or 4 ring atoms are selected from N, O, S, P, preferably from N, O or S. "4-10 membered heterocyclyl" is intended to mean a saturated, unsaturated or partially saturated ring having 4 to 10 atoms. In some embodiments, the heterocycloalkyl group can be a heterocycloalkyl group fused with an aromatic or heteroaromatic ring group. When a prefix such as 4-8 or 4-10 membered is used to represent a heterocycloalkyl group, the number of carbons is also meant to include heteroatoms. Including monocyclic, bicyclic, tricyclic, spiro, or bridged rings. Examples of heterocycloalkyl groups are: pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydropyridinyl, azetidinyl, thiazolidinyl, oxazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, azepanyl, diazepinyl, oxazepanyl, and the like. The term "heterocycloalkyl" may be used interchangeably with the term "heteroalkyl" ring.

The term "alkenyl", alone or as part of another substituent, means having at leastCarbon-carbon sp ² Straight-chain or branched monovalent hydrocarbon radicals of two to forty carbon atoms of double bonds (e.g. C ₂ -C ₆ Alkenyl radicals, also e.g. C ₂ -C ₄ Alkenyl) and includes groups having "cis" and "trans" orientations or "E" and "Z" orientations. Examples of alkenyl groups include, but are not limited to, vinyl and allyl.

The term "alkynyl", alone or as part of another substituent, refers to a straight or branched chain monovalent hydrocarbon radical of two to forty carbon atoms having at least one carbon-carbon sp triple bond (e.g., C ₂ -C ₆ Alkynyl radicals, also e.g. C ₂ -C ₄ Alkynyl). Examples of alkynyl groups include, but are not limited to, ethynyl and propynyl.

The term "alkoxy" when used alone or as part of another substituent means a group-O-R ^Q Wherein R is ^Q Is an "alkyl" group as defined above.

The term "oxo" when used alone or as part of another substituent means that the two hydrogens on the methylene group are replaced with oxygen, i.e., the methylene group is replaced with a carbonyl group, representing =o.

The term "aromatic ring" when used alone or as part of another substituent means a monocyclic or polycyclic carbocycle having 6 to 20 carbon atoms, wherein at least one ring is an aromatic ring. When one of the rings is a non-aromatic ring, the groups may be linked through an aromatic ring or through a non-aromatic ring. Examples of aryl groups include, but are not limited to: phenyl, naphthyl, tetrahydronaphthyl, 2, 3-indanyl, biphenyl, phenanthryl, anthracyl and acenaphthylenyl. The term "aromatic ring" may be used interchangeably with the term "aryl".

The term "heteroaryl ring", alone or as part of another substituent, refers to a monocyclic or polycyclic carbocyclic ring in which at least one ring atom is a heteroatom independently selected from oxygen, sulfur and nitrogen, the remaining ring atoms being C, wherein at least one ring is an aromatic ring. The group may be a carbon group or a heteroatom group (i.e., it may be C-linked or N-linked, as long as it is possible). When one of the rings is a non-aromatic ring, the groups may be linked through an aromatic ring or through a non-aromatic ring. Examples of heteroaryl groups include, but are not limited to: imidazolyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazole, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, N-methylpyrrolidinyl, and tetrahydroquinolinyl. The term "heteroaryl" may be used interchangeably with the terms "heteroaromatic", "heteroaryl" or "heteroaryl ring radical".

The term "bicyclic" refers to a group having two connecting rings, alone or as part of another substituent. A bicyclic ring may be a carbocycle (all ring atoms being carbon atoms) or a heterocycle (ring atoms include, in addition to carbon atoms, for example, 1, 2 or 3 heteroatoms, such as N, O or S). Both rings may be aliphatic (e.g., decalin and norbornane), or may be aromatic (e.g., naphthalene), or a combination of aliphatic and aromatic (e.g., tetrahydronaphthalene).

Bicyclic rings include (a) spiro compounds in which two rings share only one single atom (the spiro atom, which is typically a quaternary carbon). Examples of spiro compounds include, but are not limited to:

spirocycloalkyl groups also containing a spiro atom common to both the monocyclocycloalkyl and heterocycloalkyl groups, non-limiting examples include:

(b) Fused bicyclic compounds in which two rings share two adjacent atoms. In other words, the rings share a covalent bond, i.e. the bridgehead atoms are directly linked (e.g. α -thurene and decalin). Examples of fused bicyclic rings include, but are not limited to:

and (c) a bridged bicyclic compound, wherein the two rings share three or more atoms and the two bridgehead atoms are separated by a bridge comprising at least one atom. For example, norbornane, also known as bicyclo [2.2.1] heptane, can be considered a pair of cyclopentane rings, each sharing three of their five carbon atoms. Examples of bridged bicyclic rings include, but are not limited to:

compounds provided herein, including intermediates useful in the preparation of compounds provided herein, contain reactive functional groups (such as, but not limited to, carboxyl, hydroxyl, and amino moieties), and also include protected derivatives thereof. "protected derivatives" are those compounds in which one or more reactive sites are blocked by one or more protecting groups (also referred to as protecting groups). Suitable protecting groups for the carboxyl moiety include benzyl, t-butyl, and the like, as well as isotopes and the like. Suitable amino and amido protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable hydroxyl protecting groups include benzyl and the like. Other suitable protecting groups are well known to those of ordinary skill in the art.

In this application, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted aryl" means that the aryl group is substituted or unsubstituted, and the description includes both substituted aryl groups and unsubstituted aryl groups.

In the present application, the term "salt" or "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

By "pharmaceutically acceptable acid addition salt" is meant a salt with an inorganic or organic acid that retains the biological effectiveness of the free base without other side effects. By "pharmaceutically acceptable base addition salt" is meant a salt formed with an inorganic or organic base that is capable of maintaining the bioavailability of the free acid without other side effects. In addition to pharmaceutically acceptable salts, other salts are contemplated by the present invention. They may serve as intermediates in the purification of the compounds or in the preparation of other pharmaceutically acceptable salts or may be used in the identification, characterization or purification of the compounds of the invention.

The term "amine salt" refers to the product of neutralizing an alkyl primary, secondary or tertiary amine with an acid. The acid includes inorganic or organic acids as described herein.

The term "stereoisomer" refers to an isomer produced by the spatial arrangement of atoms in a molecule, and includes cis-trans isomers, enantiomers, non-corresponding isomers and conformational isomers.

Depending on the choice of starting materials and methods, the compounds according to the invention may be present in the form of one of the possible isomers or mixtures thereof, for example as pure optical isomers or as isomer mixtures, for example as racemic and diastereomeric mixtures, depending on the number of asymmetric carbon atoms. When describing optically active compounds, the prefix D and L or R and S are used to denote the absolute configuration of the molecule in terms of chiral center (or chiral centers) in the molecule. The prefixes D and L or (+) and (-) are symbols for designating the rotation of plane polarized light by a compound, where (-) or L represents that the compound is left-handed. The compound prefixed with (+) or D is dextrorotatory.

When the bonds to chiral carbons in the formulae of the present invention are depicted in straight lines, it is understood that both the (R) and (S) configurations of the chiral carbons and the enantiomerically pure compounds and mixtures thereof resulting therefrom are included within the general formula. The graphic representation of racemates or enantiomerically pure compounds herein is from Maehr, J.chem. Ed.1985, 62:114-120. The absolute configuration of a solid center is represented by wedge-shaped keys and dashed keys.

The term "tautomer" refers to a functional group isomer that results from the rapid movement of an atom in a molecule at two positions. The compounds of the present invention may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Proton-mobile tautomers result from the migration of a hydrogen atom covalently bonded between two atoms. Tautomers generally exist in equilibrium and attempts to isolate individual tautomers often result in a mixture whose physicochemical properties are consistent with the mixture of compounds. The location of the equilibrium depends on the chemical nature of the molecule. For example, among many aliphatic aldehydes and ketones such as acetaldehyde, the ketone type predominates; whereas, among phenols, the enol form is dominant. The present invention encompasses all tautomeric forms of the compounds.

The term "solvate" refers to a compound of the invention or a salt thereof that includes a stoichiometric or non-stoichiometric solvent that binds with non-covalent intermolecular forces, and when the solvent is water, is a hydrate.

The term "prodrug" refers to a compound of the invention that can be converted to a biologically active compound under physiological conditions or by solvolysis. Prodrugs of the invention are prepared by modifying functional groups in the compounds, which modifications may be removed by conventional procedures or in vivo to give the parent compound. Prodrugs include compounds wherein a hydroxyl group or amino group of a compound of the invention is attached to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl group, free amino group, respectively.

In this application, "pharmaceutical composition" refers to a formulation of a compound of the invention with a medium commonly accepted in the art for delivery of biologically active compounds to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote the administration of organisms, facilitate the absorption of active ingredients and further exert biological activity.

In this application, "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonizing agent, solvent, or emulsifying agent that is approved by the relevant government regulatory agency as acceptable for human or livestock use.

The term "adjuvant" refers to a pharmaceutically acceptable inert ingredient. Examples of the category of the term "excipient" include, without limitation, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients can enhance the handling characteristics of the pharmaceutical formulation, i.e., by increasing flowability and/or tackiness, making the formulation more suitable for direct compression.

The term "treatment" refers to therapeutic therapy. When specific conditions are involved, treatment refers to: (1) alleviating a disease or one or more biological manifestations of a disorder, (2) interfering with (a) one or more points in a biological cascade that results in or causes a disorder or (b) one or more biological manifestations of a disorder, (3) ameliorating one or more symptoms, effects, or side effects associated with a disorder, or one or more symptoms, effects, or side effects associated with a disorder or treatment thereof, or (4) slowing the progression of a disorder or one or more biological manifestations of a disorder.

The term "preventing" refers to a reduced risk of acquiring or developing a disease or disorder.

The term "patient" refers to any animal, preferably a mammal, that is about to or has received administration of the compound or composition according to embodiments of the present invention. The term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., with humans being preferred.

The term "therapeutically effective amount" refers to an amount of a compound that, when administered to a patient, is sufficient to effectively treat a disease or disorder described herein. The "therapeutically effective amount" will vary depending on the compound, the condition and severity thereof, and the age of the patient to be treated, and can be adjusted as desired by those skilled in the art.

The reaction temperature of each step may be appropriately selected depending on the solvent, starting material, reagent, etc., and the reaction time may be appropriately selected depending on the reaction temperature, solvent, starting material, reagent, etc. After the reaction of each step is finished, the target compound can be separated and purified from the reaction system according to a common method, such as filtration, extraction, recrystallization, washing, silica gel column chromatography and the like. Under the condition of not influencing the next reaction, the target compound can also directly enter the next reaction without separation and purification.

The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.

Advantageous effects

The present inventors have studied extensively and intensively, and have unexpectedly developed a compound or a pharmaceutically acceptable salt thereof, and a preparation method and use thereof. The invention provides a compound shown in a formula I, a tautomer, a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof, wherein the compound shown in the formula I has remarkable preventive and/or therapeutic effects on nerve cytotoxicity caused by abnormal protein such as mSOD1 aggregation, and has higher safety and pharmaceutical properties. Experiments show that the compound has a protective effect on nerve cell injury induced by abnormal proteins such as mSOD1 protein aggregation and has excellent pharmacokinetic properties.

Drawings

FIG. 1 shows the trend of the cytoprotection rate with the drug concentration (cell activity rate of added compound-cell activity rate of no added compound) in example 3, which represents that 0.05. Gtoreq.P value > 0.01; * Represents 0.01.gtoreq.P > 0.001.

Detailed Description

The invention will be further illustrated with reference to specific examples. It is to be understood that the following description is only of the most preferred embodiments of the present invention and should not be taken as limiting the scope of the invention. Upon a complete understanding of the present invention, experimental methods without specific references in the following examples, generally according to conventional conditions or according to conditions suggested by the manufacturer, may make insubstantial changes to the technical solutions of the present invention, and such changes should be considered as included in the scope of the present invention.

The application has the following definitions:

symbol or unit:

IC ₅₀ : half inhibition concentration, meaning the concentration at which half of the maximum inhibition effect is achieved

M: mol/L, for example n-butyllithium (14.56 mL,29.1mmol,2.5M in n-hexane) means an n-hexane solution of n-butyllithium at a molar concentration of 2.5mol/L

N: equivalent concentration, e.g. 2N hydrochloric acid means 2mol/L hydrochloric acid solution

Reagent:

THF: tetrahydrofuran (THF)

LiHMDS: lithium bis (trimethylsilyl) amide

DCM: dichloromethane (dichloromethane)

DEAD: azodicarboxylic acid diethyl ester

DIBAL-H: diisobutyl aluminum hydride

Example 1:5- (1- (1- (3, 5-bistrifluoromethyl) phenoxy) ethyl-2, 2-d ₃ ) Preparation of cyclohexane-1, 3-dione (Compound 1)

The synthetic route for compound 1 is as follows:

the first step: 2- (benzyloxy) propionic acid ethyl ester-3, 3-d ₃ Synthesis of (1-2)

Into a 250mL three-necked flask, compound (1-1) (10.0 g,51.5 mmol) was dissolved in anhydrous THF (100 mL) using N ₂ Atmosphere was replaced three times, then the temperature was lowered to-78℃and a THF solution of LiHMDS (78 mL,1.0mol/L,77.2 mmol) was slowly added dropwise, and after stirring at-78℃for half an hour, CD was slowly added dropwise to the reaction flask ₃ I (9.0 g,61.8 mmol) was slowly warmed to room temperature after the addition was completed and stirred for 1 hour. Slowly pouring the reaction solution into saturated NH ₄ In Cl (aq.100 mL), extracting with ethyl acetate, concentrating the organic phase, mixing, and separating and purifying with silica gel column (petroleum ether: ethyl acetate, V/V=20:1-8:1) to obtain 2- (benzyloxy) propionic acid ethyl acetateEster-3, 3-d ₃ (1-2) (4.8 g, yield 44.1%).

LC-MS,M/Z(ESI):212.2[M+H] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ ):δ7.46–7.17(m,5H),4.70(d,J＝11.6Hz,1H),4.45(d,J＝11.6Hz,1H),4.21(m,2H),4.04(s,1H),1.36–1.20(m,3H)ppm.

And a second step of: 2-Hydroxypropionic acid ethyl ester-3, 3-d ₃ Synthesis of (1-3)

In a 100mL three-necked flask, compound (1-2) (4.8 g,22.7 mmol) was dissolved in methanol (48 mL) followed by H ₂ Pd/C (10%, 960.0 mg) was added to the reaction system under an atmosphere and one drop of acetic acid was added dropwise, and the system was replaced three times under a hydrogen balloon atmosphere and stirred at room temperature for 16 hours. The reaction solution is filtered rapidly, and the filtrate is concentrated to obtain the 2-hydroxy ethyl propionate-3, 3-d ₃ (1-3) (2.1 g) was directly used in the next reaction.

LC-MS,M/Z(ESI):122.2[M+H] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ ):δ4.31–4.02(m,3H),3.18(s,1H),1.22(t,J＝7.1Hz,3H)ppm.

And a third step of: 2- (3, 5-bis (trifluoromethyl) phenoxy) propionic acid ethyl ester-3, 3-d ₃ Synthesis of (1-5)

Into a 100mL three-necked flask, compound (1-4) (2.0 g,8.7 mmol), compound (1-3) (2.1 g), PPh were placed ₃ (3.4 g,13 mmol) was dissolved in THF (20 mL) and the system was then run with N ₂ Atmosphere was changed three times, the temperature was lowered to 0℃and DEAD (3.0 g,17.4 mmol) was slowly added dropwise, and after the completion of the addition, the reaction system was slowly warmed to room temperature and stirred for 16 hours. Slowly dropping saturated NH into the reaction liquid ₄ Cl (aq.20 mL) solution is quenched, ethyl acetate is extracted, the organic phase is concentrated and dried, and then mixed, and the mixture is separated and purified by a silica gel column (petroleum ether: ethyl acetate, V/V=20:1-3:1) to obtain 2- (3, 5-bis (trifluoromethyl) phenoxy) ethyl propionate-3, 3-d ₃ (1-5) (1.3 g, 44.8% yield).

LC-MS,M/Z(ESI):334.2[M+H] ⁺

¹ H NMR(400MHz,CDCl ₃ ):δ7.48(d,J＝5.7Hz,1H),7.29(s,2H),4.82(s,1H),4.31-4.19(m,2H),1.26(t,J＝7.1Hz,3H)ppm.

Fourth step: 2- (3, 5-bis (trifluoromethyl) phenoxy) propanal-3, 3-d ₃ Synthesis of (1-6)

In a 100mL three-necked flask, compound (1-5) (1.3 g,3.9 mmol) was dissolved in DCM (13 mL) using N ₂ Atmosphere replacing three times, reducing temperature to-78deg.C, then slowly dropwise adding DIBAL-H (3.9 mL,3.9mmol,1.0mol/L in tolene) solution into the reaction system, stirring at-78deg.C for 1 hr, and adding saturated NH at the temperature ₄ The reaction was quenched with Cl (aq.20 mL), filtered, the cake washed with DCM (20 mL), the resulting mother liquor was separated, the organic phase dried over anhydrous sodium sulfate, filtered, the cake washed with DCM (20 mL), and the mother liquor was spun dry to give 2- (3, 5-bis (trifluoromethyl) phenoxy) propanal-3, 3-d ₃ (1-6) (900 mg, 79.6% yield).

LC-MS,M/Z(ESI):290.19[M+H] ⁺ 。

Fifth step: (E) -5- (3, 5-bis (trifluoromethyl) phenoxy) hex-3-en-2-one-6, 6-d ₃ Synthesis of (1-7)

In a 100mL single vial was added compound (1-6) (900 mg,3.1 mmol) dissolved in THF (9 mL), followed by 1-triphenylphosphine-2-propanone (1.3 g,4.1 mmol) and the system stirred at room temperature for 16 hours. The reaction solution was dried by spin-drying and then stirred, and purified by silica gel column separation (petroleum ether: ethyl acetate, V/v=10:1-3:1) to give (E) -5- (3, 5-bis (trifluoromethyl) phenoxy) hex-3-en-2-one-6, 6-d ₃ (1-7) (610 mg, 59.8% yield).

LC-MS,M/Z(ESI):330.26[M+H] ⁺ 。

Sixth step: 5- (1- (1- (3, 5-bistrifluoromethyl) phenoxy) ethyl-2, 2-d ₃ ) Cyclohexane-1, 3-dione (Compound 1)

In a 100mL single-necked flask, compound (1-7) (610 mg,1.9 mmol) and diethyl malonate (326.4 mg,2.0 mmol) were dissolved in EtOH (6 mL), followed by slowly adding EtONa (1.2g,3.7mmol,21%in EtOH) solution to the reaction system, and stirring the system at room temperature for 16 hours. NaOH (aq.2 mL, 2.0N) solution was added to the reaction solution, the pH was adjusted to be alkaline, and then stirred overnight, HCl (aq.6 mL, 2.0N) solution was added to the reaction solution,after the pH is regulated to be acidic, the temperature is raised to 90 ℃ and the mixture is stirred for 1 hour, H is added into the reaction system ₂ O (3 mL) and ethyl acetate (3 mL), extracting, spin-drying the organic phase, and preparing, separating and purifying (column: fimbristyliank C) ₁₈ 400 x 30mm x 10 μm, solvent: a=water: canb=acetonitrile; gradient: 0% -60%) to obtain 5- (1- (1- (3, 5-bistrifluoromethyl) phenoxy) ethyl-2, 2-d ₃ ) Cyclohexane-1, 3-dione (compound 1) (280 mg, 40.7% yield).

LC-MS,M/Z(ESI):371.9[M+H] ⁺ 。

¹ H NMR(400MHz,DMSO-d ₆ )：δ11.12(s,1H),7.61(d,J＝8.7Hz,3H),5.23(s,1H),4.73(d,J＝3.6Hz,1H),2.48–2.11(m,5H)ppm.

Example 2: preparation of (S) -5- (1- (3, 5-bis (trifluoromethyl) phenoxy-4-d) ethyl) cyclohexane-1, 3-dione (Compound 4A)

The synthetic route for compound 4A is as follows:

the first step: synthesis of 4-nitro-3, 5-bis (trifluoromethyl) phenol (4-1)

In a 100mL three-necked flask, compound (1-4) (1.0 g,4.3 mmol) was dissolved in glacial acetic acid (10 mL), followed by slow dropwise addition of fuming nitric acid (0.3 mL) and stirring at room temperature for 72 hours. The reaction solution was slowly poured into water, extracted with ethyl acetate, and the organic phase was concentrated to dryness, and then purified by column chromatography on silica gel (petroleum ether: ethyl acetate, V/v=10:1) to give 4-nitro-3, 5-bis (trifluoromethyl) phenol (4-1) (200 mg, 23.9%).

LC-MS,M/Z(ESI):276.1[M+H] ⁺ 。

And a second step of: synthesis of 5- ((4-methoxybenzyl) oxy) -2-nitro-1, 3-bis (trifluoromethyl) benzene (4-2)

In a 100mL three-necked flask, compound (4-1) (200 mg,0.7 mmol) was dissolved in acetone (10 mL), followed by addition of K to the reaction system ₂ CO ₃ (300 mg,2.1 mmol) and p-methoxybenzyl bromide (219 mg,10.5 mmol) were added dropwise and the system stirred at 60℃for 16 h. The reaction solution was rapidly filtered, the mother liquor was dried by spin-drying, and the mixture was passed through a silica gel column to give 5- ((4-methoxybenzyl) oxy) -2-nitro-1, 3-bis (trifluoromethyl) benzene (4-2) (100 mg, 34.8%).

LC-MS,M/Z(ESI):396.3[M+H] ⁺ 。

And a third step of: synthesis of 4- ((4-methoxybenzyl) oxy) -2, 6-bis (trifluoromethyl) aniline (4-3)

In a 100.0mL three-necked flask, compound (4-2) (100 mg,0.3 mmol) was dissolved in methanol (5 mL), then Raney nickel (10%, 960.0 mg) was added to the reaction system, the system was replaced three times under a hydrogen balloon atmosphere, and the reaction was completed by stirring at room temperature for 16 hours, and a new target point was formed. The reaction solution was rapidly filtered and the mother liquor was dried to give 4- ((4-methoxybenzyl) oxy) -2, 6-bis (trifluoromethyl) aniline (4-3) (100 mg, 100%).

LC-MS,M/Z(ESI):366.3[M+H] ⁺ 。

Fourth step: synthesis of 2-bromo-5- ((4-methoxybenzyl) oxy) -1, 3-bis (trifluoromethyl) benzene (4-4)

In a 100mL three-necked flask, compound (4-3) (750 mg,1.7 mmol) was dissolved in acetonitrile (20 mL), followed by addition of cuprous bromide (0.5 g,3.4 mmol) to the reaction system, followed by dropwise addition of isoamyl nitrite (307 mg,2.6 mmol), and the system was stirred at room temperature for 16 hours to complete the reaction. The reaction solution was rapidly filtered, and the mother liquor was dried by spin-drying, and passed through a silica gel column to give 2-bromo-5- ((4-methoxybenzyl) oxy) -1, 3-bis (trifluoromethyl) benzene (4-4) (800 mg, 90.8%).

LC-MS,M/Z(ESI):430.2[M+H] ⁺ 。

Fifth step: synthesis of 1- ((4-methoxybenzyl) oxy) -3, 5-bis (trifluoromethyl) benzene-4-deuterium (4-5)

In a 100mL three-necked flask, compound (4-4) (800 mg,22.7 mmol) was dissolved in deuterated acetonitrile (20 mL), and then hexamethyldisilane (546 mg,45 mmol) and then potassium methoxide (261mg, 45 mmol) were added to the reaction system, and the system was stirred under nitrogen atmosphere at room temperature for 16 hours to complete the reaction. The reaction solution was rapidly filtered and the mother liquor was dried to give 1- ((4-methoxybenzyl) oxy) -3, 5-bis (trifluoromethyl) benzene-4-deuterium (4-5) (250 mg, 38.2%).

LC-MS,M/Z(ESI):352.3[M+H] ⁺ 。

Sixth step: synthesis of 3, 5-bis (trifluoromethyl) phenol-4-deuterium-alcohol (4-6)

In a 100.0mL three-necked flask, compound (4-5) (250 mg,0.7 mmol) was dissolved in methylene chloride (5 mL), followed by adding trifluoroacetic acid (5 mL) to the reaction system, and the system was stirred at room temperature for 16 hours. The reaction solution was dried by spin-drying and then directly used in the next reaction to give 3, 5-bis (trifluoromethyl) phenol-4-deuterium-alcohol (4-6) (200 mg).

LC-MS,M/Z(ESI):232.1[M+H] ⁺ 。

Seventh step: synthesis of ethyl (S) -2- (3, 5-bis (trifluoromethyl) phenoxy-4-deuterium) propionate (4-7)

In a 100mL three-necked flask, compound (4-6) (200 mg,0.9 mmol), (R) -ethyl lactate (200 mg, crude product), PPh ₃ (340 mg,1.3 mmol) was dissolved in tetrahydrofuran (20 mL), followed by N ₂ Atmosphere was changed three times, the temperature was lowered to 0 ℃, and diethyl azodicarboxylate (300 mg,1.7 mmol) was slowly added dropwise, and after the addition was completed, the reaction system was slowly warmed to room temperature and stirred for 16 hours. Slowly dropping saturated NH into the reaction liquid ₄ Cl (aq.20.0 mL) solution, ethyl acetate extraction, liquid separation, organic phase concentration, sample mixing, and silica gel column separation and purification (petroleum ether: ethyl acetate, V/V=20:1-3:1) to obtain (S) -2- (3, 5-bis (trifluoromethyl) phenoxy-4-deuterium) ethyl propionate (4-7) (100 mg, 34.9%).

LC-MS,M/Z(ESI):332.2[M+H] ⁺ 。

Eighth step: (S) -2- (3, 5-bis (trifluoromethyl) phenoxy-4-deuterium) propanol (4-8)

In a 100.0mL three-necked flask, compound (4-7) (100 mg,0.3 mmol) was dissolved in THF (10 mL) using N ₂ Atmosphere was replaced three times, the temperature was lowered to 0℃and DIBAl-H (3.9 mL,3.9mmol of toluene solution) was slowly added dropwise to the reaction system, and after the addition was completed, the system was kept stirring at 0℃for 1 hour, and then saturated NH was added at this temperature ₄ The reaction was quenched with Cl (aq.20 mL), filtered, the filter cake washed with dichloromethane (20 mL), the resulting mother liquor was partitioned,the organic phase was dried over anhydrous sodium sulfate, filtered, and the filter cake was washed with dichloromethane (20 mL) and the mother liquor was spin-dried to give (S) -2- (3, 5-bis (trifluoromethyl) phenoxy-4-deuterium) propanol (4-8) (100 mg, 79.6%).

LC-MS,M/Z(ESI):290.2[M+H] ⁺ 。

Ninth step: synthesis of (S) -2- (3, 5-bis (trifluoromethyl) phenoxy-4-deuterium) propanal (4-9)

In a 100.0mL three-necked flask, compound (4-8) (100 mg,0.3 mmol) was dissolved in methylene chloride (10 mL), then, dessmartin reagent (292 mg,0.7 mmol) was added, and after the addition, the system was kept under stirring at 0℃for 2 hours, after which saturated NH was added at this temperature ₄ The reaction was quenched with Cl (aq.20 mL), filtered, the filter cake washed with dichloromethane (20 mL), the resulting mother liquor was partitioned, the organic phase dried over anhydrous sodium sulfate, filtered, the filter cake washed with dichloromethane (20 mL) and the mother liquor was spun dry to give (S) -2- (3, 5-bis (trifluoromethyl) phenoxy-4-deuterium) propanal (4-9) (100 mg, 100%).

LC-MS,M/Z(ESI):288.2[M+H] ⁺ 。

Tenth step: synthesis of (S, E) -5- (3, 5-bis (trifluoromethyl) phenoxy-4-deuterium) hex-3-en-2-one (4-10)

In a 100mL single-necked flask, the compound (4-9) (100 mg,0.3 mmol) was dissolved in tetrahydrofuran (10 mL), followed by 1-triphenylphosphine-2-propanone (130 mg,0.4 mmol) and the system was stirred at room temperature for 16 hours. The reaction mixture was dried by spin-drying, and then stirred and purified by silica gel column (petroleum ether: ethyl acetate, V/v=10:1-3:1) to give (S, E) -5- (3, 5-bis (trifluoromethyl) phenoxy-4-deuterium) hex-3-en-2-one (4-10) (100.0 mg, 87.6%).

LC-MS,M/Z(ESI):328.2[M+H] ⁺ 。

Eleventh step: synthesis of (S) -5- (1- (3, 5-bis (trifluoromethyl) phenoxy-4-deuterium) ethyl) cyclohexane-1, 3-dione (4A)

In a 100mL single-necked flask, the compound (4-10) (100 mg,0.3 mmol) and diethyl malonate (326.4 mg,2.0 mmol) were dissolved in ethanol (6 mL), followed by slowly adding sodium ethoxide (1.2 g,3.7mmol,21% ethanol solution) to the reaction system, and stirring the system at room temperature for 16 hours. NaOH is added into the reaction solution(aq.2 mL, 2.0N) solution, adjusting pH to alkaline, stirring overnight, adding HCl (aq.6 mL, 2.0N) solution to the reaction solution, adjusting pH to acidity, heating the system to 90deg.C, stirring for 1 hr, adding H to the reaction system ₂ O (3 mL) and ethyl acetate (3 mL), extracting, spin-drying the organic phase, and performing preparation, separation and purification (column: fimbristyliank C) ₁₈ 400 x 30mm x 10 μm, solvent: a=water, b=acetonitrile; gradient: 0% -60%) to give (S) -5- (1- (3, 5-bis (trifluoromethyl) phenoxy-4-deuterium) ethyl) cyclohexane-1, 3-dione (compound 4A) (41 mg, 36.3%).

LC-MS,M/Z(ESI):368.1[M-H] ^- 。

¹ H NMR(400MHz,DMSO-d ₆ )δ11.18(s,1H),7.61(s,2H),5.21(s,1H),4.72(d,1H),2.48–2.11(m,5H),1.23(d,3H)ppm.

Example 3: preparation of 5- (1- (3, 5-bis (trifluoromethyl) phenoxy) ethyl-1-deuterium) cyclohexane-1, 3-dione (Compound 5)

The synthetic route for compound 5 is as follows:

the first step: synthesis of (1S, 3R,4R, 5R) -1,3, 4-trihydroxy-6-oxabicyclo [3.2.1] oct-7-one (5-2)

To a suspension of (1S, 3R,4S, 5R) -1,3,4, 5-tetrahydroxycyclohexane-1-carboxylic acid (5-1) (7.6 g,46.8 mmol) in toluene (140 mL) under nitrogen protection was added p-toluenesulfonic acid (161 mg,0.94 mmol) and then heated to 120℃for reaction for 24 hours. The reaction solution was cooled to room temperature, toluene as an organic solvent was dried by spinning, and the crude product was dissolved with acetone, filtered, and the filtrate was dried by spinning to give (1S, 3R,4R, 5R) -1,3, 4-trihydroxy-6-oxabicyclo [3.2.1] oct-7-one (5-2) (3.6 g, yield: 44.6%).

LC-MS,M/Z(ESI):175.05[M+H] ⁺ 。

¹ H NMR(400MHz,MD ₃ OD)δ4.75(dd,1H),4.04(dd,1H),3.75(m,1H),2.51(d,1H),2.26–2.18(m,1H),2.06–1.99(m,1H),1.97(d,1H)ppm.

And a second step of: synthesis of (1R, 3R,4S, 5R) -3- ((tert-butyldimethylsilyl) oxy) -1, 4-dihydroxy-6-oxabicyclo [3.2.1] octan-7-one (5-3)

(1S, 3R,4R, 5R) -1,3, 4-trihydroxy-6-oxabicyclo [3.2.1] oct-7-one (5-2) (3.6 g,20.6 mmol), imidazole (2.12 g,31 mmol) and t-butyldimethylchlorosilane (4.05 g,26.9 mmol) were dissolved in anhydrous DMF (100 mL) under nitrogen protection and then reacted at room temperature for 16 hours. The reaction was quenched with water, extracted with ethyl acetate, the organic phase concentrated to dryness and stirred, and purified by column chromatography on silica gel (petroleum ether: ethyl acetate, V/v=5:1 to 1:1) to give (1 r,3r,4s,5 r) -3- ((tert-butyldimethylsilyl) oxy) -1, 4-dihydroxy-6-oxabicyclo [3.2.1] octan-7-one (5-3) (3.8 g, yield: 65%)

LC-MS,M/Z(ESI):289.14[M+H] ⁺ 。

And a third step of: synthesis of O, O '- ((1R, 3R,4S, 5R) -3- ((tert-butyldimethylsilyl) oxy) -7-oxo-6-oxabicyclo [3.2.1] octane-1, 4-diyl) O, O' -diphenyldisulfate (5-4)

To a solution of (1R, 3R,4S, 5R) -3- ((tert-butyldimethylsilyl) oxy) -1, 4-dihydroxy-6-oxabicyclo [3.2.1] octan-7-one (5-3) (3.8 g,13.1 mmol) and 4-dimethylaminopyridine (4.8 g,39.3 mmol) in methylene chloride (130 mL) under nitrogen was added triethylamine (9.1 mL) and phenyl thiochloroformate (4.5 mL,26.2 mmol). Then reacted at room temperature for 2 hours. The reaction was quenched with water, extracted with ethyl acetate, the organic phase concentrated to dryness and stirred, and purified by column chromatography on silica gel (petroleum ether: ethyl acetate, V/v=5:1 to 2:1) to give O, O '- ((1 r,3r,4s,5 r) -3- ((tert-butyldimethylsilyl) oxy) -7-oxo-6-oxabicyclo [3.2.1] octane-1, 4-diyl) O, O' -diphenyl disulfate (5-4) (4.2 g, yield: 57%)

LC-MS,M/Z(ESI):561.14[M+H] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ )δ7.32(t,4H),7.20(d,2H),7.15–7.07(m,4H),5.86(t,1H),5.10(t,1H),4.29–4.21(m,1H),3.81–3.74(m,1H)2.64(d,1H),2.53–2.39(m,2H),0.92(s,9H),0.12(s,6H)ppm.

Fourth step: synthesis of (1S, 3R, 5R) -3- ((tert-butyldimethylsilyl) oxy) -6-oxabicyclo [3.2.1] octan-7-one (5-5)

O, O '- ((1R, 3R,4S, 5R) -3- ((tert-butyldimethylsilyl) oxy) -7-oxo-6-oxabicyclo [3.2.1] octane-1, 4-diyl) O, O' -diphenyldisulfate (5-4) (4.2 g,7.5 mmol), tributyltin hydride (16 g,55 mmol) and azobisisobutyronitrile (1.72 g,10.5 mmol) in dry toluene (250 mL) were heated to 120℃under nitrogen protection, the reaction mixture was cooled to room temperature, and the crude product was concentrated and purified by column chromatography on silica gel (petroleum ether: ethyl acetate, V/V=50:1 to 5:1) to give (1S, 3R, 5R) -3- ((tert-butyldimethylsilyl) oxy) -6-oxabicyclo [3.2.1] octane-7-one (5-5) (1.2 g, 62.5%)

LC-MS,M/Z(ESI):257.15[M+H] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ )δ4.87(t,1H),3.97(t,1H),3.93–3.87(m,1H),2.94(s,1H),2.63(t,2H),2.32–2.25(m,1H),2.07–1.93(m,2H)0.90(s,9H),0.09(s,6H)ppm.

Fifth step: synthesis of (1S, 3R, 5R) -3- ((tert-butyldimethylsilyl) oxy) -5-hydroxy-N-methoxy-N-methylcyclohexane-1-carboxamide (5-6)

To a solution of (1S, 3R, 5R) -3- ((tert-butyldimethylsilyl) oxy) -6-oxabicyclo [3.2.1] octan-7-one (5-5) (1.2 g,4.68 mmol) and N-methyl-N-methoxyamine hydrochloride (686 mg,7 mmol) in tetrahydrofuran (25 mL) at 0deg.C under nitrogen was added dropwise a magnesium isopropylchloride format reagent (1.3M tetrahydrofuran solution, 10.8 mL). Then reacted at room temperature for 2 hours, quenched with water under ice bath condition, extracted with ethyl acetate, and the organic phase concentrated to dryness and stirred, and purified by column chromatography on silica gel (petroleum ether: ethyl acetate, V/v=5:1 to 2:1) to give (1 s,3r,5 r) -3- ((tert-butyldimethylsilyl) oxy) -5-hydroxy-N-methoxy-N-methylcyclohexane-1-carboxamide (5-6) (818 mg, yield: 55%)

LC-MS,M/Z(ESI):318.20[M+H] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ )δ4.26–4.21(m,1H),4.15–4.01(m,1H),3.67(s,3H),3.34–3.21(m,1H),3.17(s,3H),2.05–1.94(m,2H),1.75–1.65(m,1H)1.63–1.35(m,3H),0.90(s,9H),0.09(s,6H)ppm.

Sixth step: synthesis of (3R, 5R) -3, 5-bis ((t-butyldimethylsilyl) oxy) -N-methoxy-N-methylcyclohexane-1-carboxamide (5-7)

(1S, 3R, 5R) -3- ((tert-Butyldimethylsilyl) oxy) -5-hydroxy-N-methoxy-N-methylcyclohexane-1-carboxamide (5-6) (818 mg,2.58 mmol), imidazole (264 mg,3.87 mmol) and tert-Butyldimethylchlorosilane (506 mg,3.35 mmol) were dissolved in anhydrous DMF (26 mL) under nitrogen protection and then reacted at room temperature for 16 hours. The reaction was quenched with water, extracted with ethyl acetate, and the organic phase was concentrated to dryness, and then stirred, and purified by column chromatography on silica gel (petroleum ether: ethyl acetate, V/v=5:1 to 2:1) to give (3 r,5 r) -3, 5-bis ((t-butyldimethylsilyl) oxy) -N-methoxy-N-methylcyclohexane-1-carboxamide (5-7) (1.0 g, yield: 90%)

LC-MS,M/Z(ESI):432.29[M+H] ⁺ 。

Seventh step: synthesis of (3R, 5R) -3, 5-bis ((t-butyldimethylsilyl) oxy) cyclohexane-1-carbaldehyde-deuterium (5-8)

A solution of (3R, 5R) -3, 5-bis ((tert-butyldimethylsilyl) oxy) -N-methoxy-N-methylcyclohexane-1-carboxamide (5-7) (1.0 g,2.3 mmol) in tetrahydrofuran (5 mL) was added dropwise to a suspension of deuterated lithium aluminum hydride (200 mg,4.6 mmol) in tetrahydrofuran (5 mL) under nitrogen protection at 0deg.C, followed by reaction at 0deg.C for 2 hours, quenching the reaction with ice-water slowly, adjusting the pH of the mixture to neutral with 2M hydrochloric acid, extracting with ethyl acetate, concentrating the organic phase to dryness, and then mixing the mixture with a silica gel column, and separating and purifying (petroleum ether: ethyl acetate, V/V=5:1) with a silica gel column to obtain (3R, 5R) -3, 5-bis ((tert-butyldimethylsilyl) oxy) cyclohexane-1-carbaldehyde-deuterium (5-8) (826 mg, 95%)

LC-MS,M/Z(ESI):374.26[M+H] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ )δ4.22–4.16(m,1H),4.15–4.07(m,1H),2.72–2.62(m,1H),2.06–1.98(m,1H),1.79–1.63(m,3H),1.54–1.43(m,2H),0.88(s,9H)0.87(s,9H),0.07–0.4(m,12H)ppm.

Eighth step: synthesis of 1- ((3R, 5R) -3, 5-bis ((t-butyldimethylsilyl) oxy) cyclohexyl) ethan-1-deuterium-1-ol (5-9)

To 1- ((3R, 5R) -3, 5-bis ((tert-butyldimethylsilyl) oxy) cyclohexyl) ethan-1-deuterium-1-ol (5-9) (500 mg,1.33 mmol) in tetrahydrofuran (7 mL) was added dropwise methyl magnesium chloride format reagent (3M, 0.67 mL) under nitrogen protection at 0deg.C, reacted for 2 hours at 0deg.C, quenched with ice water, extracted with ethyl acetate, the organic phase concentrated dry and stirred, and purified (petroleum ether: ethyl acetate, V/V=5:1) by column chromatography on silica gel to give 1- ((3R, 5R) -3, 5-bis ((tert-butyldimethylsilyl) oxy) cyclohexyl) ethan-1-deuterium-1-ol (5-9) (400 mg, yield: 77%)

LC-MS,M/Z(ESI):390.29[M+H] ⁺ 。

Ninth step: synthesis of (5- (1- (3, 5-bis (trifluoromethyl) phenoxy) ethyl-1-deuterium) cyclohexane-1, 3-diyl) bis (oxy)) bis (t-butyldimethylsilane) (5-10)

Diethyl azodicarboxylate (207 mg,1.02 mmol) was added dropwise to a solution of 1- ((3R, 5R) -3, 5-bis ((tert-butyldimethylsilyl) oxy) cyclohexyl) ethan-1-deuterium-1-ol (5-9) (200 mg,0.51 mmol), 3, 5-bistrifluoromethylphenol (120 mg,0.51 mmol) and triphenylphosphine (268 mg,1.02 mmol) in tetrahydrofuran (2.5 mL) under nitrogen at 0deg.C, followed by reaction at room temperature for 16 hours. The reaction mixture was quenched with water, extracted with ethyl acetate, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and applied to a silica gel column (petroleum ether: ethyl acetate, V/v=100:1 to 10:1) to give ((5- (1- (3, 5-bis (trifluoromethyl) phenoxy) ethyl-1-deuterium) cyclohexane-1, 3-diyl) bis (oxy)) bis (t-butyldimethylsilane) (5-10) (270 mg, yield 88%).

LC-MS,M/Z(ESI):602.30[M+H] ⁺ 。

Tenth step: synthesis of 5- (1- (3, 5-bis (trifluoromethyl) phenoxy) ethyl-1 deuterium) cyclohexane-1, 3-diol (5-11)

To a solution of ((5- (1- (3, 5-bis (trifluoromethyl) phenoxy) ethyl-1-deuterium) cyclohexane-1, 3-diyl) bis (oxy)) bis (t-butyldimethylsilane) (5-10) (100 mg,0.17 mmol) in dichloromethane (2 mL) under nitrogen at 0 ℃ was added a solution of 1,4 dioxane (4 m,2 mL) of hydrochloric acid. Then reacting at 0 ℃ for 2 hours, adding water to quench the reaction, extracting by using ethyl acetate, concentrating the organic phase, mixing the organic phase with a sample, and separating and purifying by using a silica gel column (petroleum ether: ethyl acetate, V/V=10:1 to 1:2) to obtain 5- (1- (3, 5-bis (trifluoromethyl) phenoxy) ethyl-1 deuterium) cyclohexane-1, 3-diol (5-11) (40 mg, yield: 64%)

LC-MS,M/Z(ESI):374.12[M+H] ⁺ 。

Eleventh step: synthesis of 5- (1- (3, 5-bis (trifluoromethyl) phenoxy) ethyl-1-deuterium) cyclohexane-1, 3-dione (Compound 5)

To a solution of 5- (1- (3, 5-bis (trifluoromethyl) phenoxy) ethyl-1 deuterium) cyclohexane-1, 3-diol (5-11) (40 mg,0.11 mmol) in methylene chloride (2 mL) under nitrogen protection was added pyridinium chlorochromate (88 mg,0.41 mmol), then reacted at room temperature for 3 hours, quenched with water, extracted with ethyl acetate, concentrated and dried organic phase, stirred, and purified by silica gel column separation (petroleum ether: ethyl acetate, V/V=10:1 to 1:2) to give 5- (1- (3, 5-bis (trifluoromethyl) phenoxy) ethyl-1-deuterium) cyclohexane-1, 3-dione (compound 5) (5 mg, yield: 10%)

LC-MS,M/Z(ESI):370.09[M+H] ⁺ 。

¹ H NMR(400MHz,DMSO-d6)δ11.12(s,1H),7.62(s,2H),7.59(s,1H),5.23(s,1H),2.47–2.15(m,5H),1.26(s,3H),1.24(s,3H)ppm.

The compounds shown in table 1 below were prepared:

TABLE 1

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Test example 1 Compounds for SOD1-G93A transient PC-12 cytoprotection assay

PC12 cells are transfected by SOD1-G93A plasmid, 600mg/L G418 is added for screening and culturing for 2 days, the cells are collected and planted in 96-well plates, 4000 cells are planted in each well, medicines with different concentrations are added for continuous culturing for 72 hours, and CellTiter-Glo reagent is added for detecting the activity of the cells.

The compound disclosed in the application has better protection effect on SOD1-G93A transient PC-12 cells.

Test example 2: compound for detecting SOD1-G93A stable transfer PC-12 cytoprotection

PC12-SOD1-G93A cells were cultured in RPMI-1640 medium containing inactivated 5% fetal bovine serum, 100U/mL penicillin, 100. Mu.g/mL streptomycin and 1mg/mL G418 in an incubator at 37℃with 5% CO2, and the cell confluency rate reached 70-80% passage, and then sub-flask passages were performed. Cells in the logarithmic growth phase were subjected to experiments. Doxycycline induces SOD1-G93A expression. Cells in log phase were first transferred to T75 flasks and Doxycycline was added at a final concentration of 4 μg/mL and the Doxycycline was treated for 4 days. PC12-SOD1-G93A cells treated with Doxycycline for 4 days were digested and plated in 96-well plates with about 2000 cells per well, serum concentration adjusted to 1%, containing 4. Mu.g/mL Doxycycline, and cultured overnight. Compound 1 and compound 2 were diluted in multiple ratios and then added to 96-well plates and compound pretreated for 24 hours. L-GLU was diluted to 1% FBS in RPMI-1640 medium, and then added to a 96-well plate to give a final concentration of L-GLU of 20mM, and the treatment was continued for 24 hours. Cell viability was measured by adding CellTiter-Glo reagent.

The compounds disclosed herein are tested according to the methods described above and the test results demonstrate that the compounds of the present invention have a protective effect against abnormal proteins such as the aggregation-induced neuronal cell damage of the mSOD1 protein.

Test example 3: protection assay of compounds against MG 132-induced SOD1-G93A stably transformed PC-12 cell death

With RPMI-1640 medium containing inactivated 5% fetal bovine serum, 100U/mL penicillin, 100. Mu.g/mL streptomycin and 1mg/mL G418 at 37℃with 5% CO ₂ PC12-SOD1-G93A cells are cultured in the incubator, and the cell confluence rate reaches 70-80% for passage, and then the cells are subjected to bottle division and passage. Cells in the logarithmic growth phase were subjected to experiments. Doxycycline induces SOD1-G93A expression. Firstly, transferring cells in logarithmic phase into T75 cell bottle, changing culture medium into DMEM, adding Doxycycline, and finallyThe concentration was 2. Mu.g/mL and Doxycycline was treated for 4 days. PC12-SOD1-G93A cells treated with Doxycycline for 4 days were digested and plated in 96-well plates with about 2000 cells per well, serum concentration was adjusted to 2%, and cultured overnight with 2ug/mL Doxycycline. Compound NU-9 and compound 1 were subjected to double dilution and then added to a 96-well plate, and the compounds were pretreated for 24 hours. MG132 was diluted to DMEM medium with 2% FBS, and then added to 96-well plates to give a final MG132 concentration of 300nM, and the treatment was continued for 48 hours. Cell viability was measured by adding CellTiter-Glo reagent.

The compounds disclosed herein were tested according to the methods above and presented with the cytoprotective rates (compound added cell viability-compound not added cell viability) indicated in table 2 below (as shown in fig. 1).

TABLE 2

The test results show that the cytoprotection rate of the compound 1 is higher than that of NU-9 under the same concentration of the compound 1 and NU-9. The compound has remarkable cytoprotective effect on nerve cell injury induced by abnormal proteins such as mSOD1 protein aggregation.

Test example 4: human liver microsome stability

The human liver microsome stability of the control compound and the compound of the present invention was determined according to the following experimental method. The hepatic microsome stability test of the compound was tested using in vitro co-incubation of the compound with human hepatic microsomes. Test compounds were first formulated as a 10mM stock solution in DMSO solvent, followed by dilution of the compounds to 0.5mM using acetonitrile. Liver microsomes (Corning) were diluted with PBS to a microsome/buffer solution, and 0.5mM of the compound was diluted with the solution to give a working solution having a compound concentration of 1.5. Mu.M and a liver microsome concentration of 0.75mg/mL. The reaction was started by taking a deep well plate, adding 30. Mu.L of working solution per well, then adding 15. Mu.L of pre-warmed 6mM NADPH solution, and incubating at 37 ℃. At 0, 5, 15, 30, 45 minutes of incubation, the reaction was terminated by adding 135 μl acetonitrile to the corresponding wells. At the last 45 minutes After the reaction was terminated with acetonitrile at the clock time point, the deep-well plate was vortexed and vibrated for 10 minutes (600 rpm/min) and then centrifuged for 15 minutes. Taking supernatant after centrifugation, adding purified water 1:1, performing LC-MS/MS detection to obtain the ratio of the peak area of the compound to the internal standard peak area at each time point, comparing the ratio of the peak area of the compound at 5, 15, 30 and 45 minutes with the ratio of the peak area at 0 minute, calculating the residual percentage of the compound at each time point, and calculating T by using Graphpad 5 software _1/2 。

The test result shows that the compound of the invention has better stability of human liver microsome.

Test example 5: mouse pharmacokinetics

3 male CD-1 mice were used at a dose of 10mg/kg, the route of administration was gastric lavage, the vehicle was 5% DMSO+10% Solutol+85% Saline, and the blood collection time points were 15, 30 minutes and 1, 2, 4, 6, 8, 24 hours before and after administration. Blood samples 6800g were centrifuged at 2-8deg.C for 6 minutes, and plasma was collected and stored at-80deg.C. 10. Mu.L of plasma at each time point was taken, 200. Mu.L of methanol containing 100ng/mL of internal standard was added, and after vortexing and mixing, 18000g was centrifuged for 7 minutes at 2-8 ℃. 200 μl was transferred to a 96-well sample plate for quantitative LC-MS/MS analysis. The principal pharmacokinetic parameters were analyzed using the winnonlin7.0 software non-compartmental model.

The test results show that the compounds of the invention have excellent pharmacokinetic properties.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof:

wherein,

l is selected from unsubstituted or substituted by at least one R ¹ Substituted C _1-6 Alkylene or C _1-6 A halogenated alkylene group; the R is ¹ Selected from hydrogen, deuterium, C _1-6 Deuterated alkyl;

m is 0, 1, 2, 3 or 4;

n is 1, 2, 3, 4 or 5; and, in addition, the processing unit,

2. A compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof, according to claim 1, wherein the 3-10 membered heterocycloalkyl is monocyclic, fused bicyclic, bicyclic including bridged or spiro rings;

and/or the 3-10 membered heterocycloalkyl further has 1 to 3 heteroatoms selected from N, O, S;

and/or, the R ¹ Selected from deuterium and C _1-6 Deuterated alkyl.

3. A compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof according to claim 1, wherein the compound is selected from the group consisting of the following structures:

Wherein,

l is selected from unsubstituted or substituted by 1 or 2R ¹ Substituted C _1-6 Alkyl or C _1-6 A haloalkyl group; the R is ¹ Selected from deuterium, C _1-6 Deuterated alkyl;

R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ and R is ⁸ Each independently selected from hydrogen, deuterium, halogen, hydroxy, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 Alkyl, C _3-8 Cycloalkyl, C _1-6 Deuterated alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, C _3-8 Halogenated cycloalkyl, C _1-6 Alkyl hydroxy, C _1-6 Alkylcarbonyl, C _1-6 Alkoxy, C _1-6 Haloalkoxy, 3-10 membered heterocycloalkyl; the C is _1-6 Alkyl, C _3-8 Cycloalkyl, C _1-6 Deuterated alkyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, C _3-8 Halogenated cycloalkyl, C _1-6 Alkyl hydroxy, C _1-6 Alkylcarbonyl, C _1-6 Alkoxy, C _1-6 Haloalkoxy groups are optionally substituted with one or more R ^c Substitution;

the R is ^c A substituent selected from the group consisting of: halogen, hydroxy, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 Alkyl, C _1-6 Deuterated alkyl, C _2-6 Alkynyl, C _1-6 Haloalkyl, C _1-6 Alkyl hydroxy, C _1-6 Alkylcarbonyl, C _1-6 Alkoxy, C _1-6 Haloalkoxy groups; when the substituents are plural, the R ^c The same or different; and, in addition, the processing unit,

when deuterium substitution is not present on L, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ At least one of them is selected from deuterium or C _1-6 Deuterated alkyl.

4. A compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof according to claim 3,

The L is R1 or 2 ¹ Substituted C _1-6 Alkyl, R ¹ Selected from deuterium or C _1-6 Deuterated alkyl; preferably, said R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ Is H;

or, the L is unsubstituted C _1-6 Alkyl, said R ⁷ Deuterium, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ And R is ⁸ H.

5. A compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof according to claim 3, wherein the compound is selected from the group consisting of the structures:

wherein R is ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ Having the definition as defined in claim 3;

R ^1a 、R ^1b each independently selected from hydrogen, deuterium, C _1-6 Alkyl or C _1-6 Deuterated alkyl;

and R is ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ 、R ⁸ 、R ^1a And R is ^1b At least one of which contains deuterium;

preferably, said R ^1a 、R ^1b Each independently selected from hydrogen, deuterium, methyl or deuterated methyl;

preferably, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ 、R ⁸ 、R ^1a And R is ^1b At least one of which is deuterium, or deuterated methyl;

more preferably, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b Each independently is hydrogen or deuterium, and R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b At least one of them is deuterium; r is R ⁶ 、R ⁷ 、R ⁸ 、R ^1b Is hydrogen; r is R ^1a Is methyl;

more preferably, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b Is hydrogen; r is R ^1b Is hydrogen; r is R ^1a Is methyl; r is R ⁶ 、R ⁷ 、R ⁸ Each independently is hydrogenOr deuterium, and R ⁶ 、R ⁷ 、R ⁸ At least one of them is deuterium;

more preferably, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ 、R ⁸ Is hydrogen; r is R ^1a 、R ^1b Each independently selected from hydrogen, deuterium, methyl or deuterated methyl, and R ^1a 、R ^1b At least one of which is deuterium or deuterated methyl.

6. The compound of formula I, tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof according to claim 5,

the R is ^1a And R is ^1b Each independently selected from hydrogen or C _1-6 Alkyl, said R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ Each independently selected from hydrogen or deuterium, and R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ At least one of which is selected from deuterium; preferably, R ⁷ Deuterium, R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ And R is ⁸ H.

7. A compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof according to claim 3, wherein R ^2a 、R ^2b 、R ^3a 、R ^3b 、R ⁴ 、R ^5a 、R ^5b 、R ⁶ 、R ⁷ And R is ⁸ Each independently selected from hydrogen, deuterium, halogen, C _1-6 Alkyl, C _1-6 Deuterated alkyl or C _1-6 A haloalkyl group.

8. A compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof according to claim 1 or 3, wherein L is selected from the group consisting of-CH (CH ₃ )-、-CD(CH ₃ )-、-CH(CD ₃ ) -or-CD (CD) ₃ )-。

9. The compound of formula I, tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof according to claim 1, wherein R ^a And R is ^b Each independently selected from deuterium, deuterated methyl, or trifluoromethyl.

10. A compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof according to claim 1, wherein the compound comprises:

11. a compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof according to claim 1, wherein the compound comprises:

12. a pharmaceutical composition, the pharmaceutical composition comprising: a compound of formula I as defined in any one of claims 1 to 11, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof; and a pharmaceutically acceptable carrier.

13. Use of a compound of formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof, according to any one of claims 1 to 11, or a pharmaceutical composition according to claim 11, the use comprising:

preventing and/or treating diseases associated with the presence of protein folding, misfolding, or abnormal protein aggregates;

and/or preventing and/or treating neurodegenerative diseases;

And/or preventing and/or treating a disease associated with degeneration of upper motor neurons;

and/or preparing a medicament, pharmaceutical composition or formulation for the presence of protein folding, misfolding, or abnormal protein aggregate related diseases;

and/or preparing a medicament, pharmaceutical composition or formulation for the neurodegenerative disease;

and/or preparing a medicament, a pharmaceutical composition or a preparation for preventing and/or treating diseases related to the degeneration of upper motor neurons.

14. The use of claim 13, wherein the abnormal protein aggregates comprise SOD1 protein aggregates or TDP-43 protein aggregates;

and/or, the neurodegenerative disease comprises: parkinson's disease, diffuse lewy body disease, multiple system atrophy and pantothenate kinase-associated neurodegeneration, huntington's disease, jack's disease, alzheimer's disease or frontotemporal lobar degeneration;

and/or, the disease associated with degeneration of upper motor neurons comprises: amyotrophic lateral sclerosis, primary lateral sclerosis, and hereditary spastic paraplegia.

15. The use of claim 14, wherein the SOD1 protein aggregate is a G93A SOD1 protein aggregate or a G85R SOD1 protein aggregate.