Compounds for treating diseases related to mitochondrial dysfunction

Abstract

The invention relates to the field of biological medicine, and in particular relates to a compound shown in a formula (I), pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof, and application of the compound in preparation of drugs for preventing or treating mitochondrial dysfunction related diseases, such as mitochondrial dysfunction related diseases, mitochondrial dysfunction related diseases, mitochondrial dysfunction related diseases and mitochondrial dysfunction related diseases. The invention also relates to application of the compound in medicines for treating neurodegenerative diseases related to mitochondrial dysfunction. The compounds of formula (I) are capable of binding mitochondria to LC3B.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to a compound of formula (I), a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof, and use thereof in the preparation of a drug for preventing or treating diseases associated with mitochondrial dysfunction, in particular neurodegenerative diseases associated with mitochondrial dysfunction. Background Art

[0002] Mitochondrial dysfunction can lead to a series of cellular abnormalities, including reduced ATP production, increased reactive oxygen species (ROS) production, and altered calcium signaling. Mitochondrial autophagy, as known in the prior art, belongs to a receptor-based selective autophagy pathway, in which damaged or depolarized mitochondria are engulfed by LC3-conjugated autophagosomes through the action of autophagy receptors and degraded by lysosomes. Mitochondrial dysfunction or impaired mitophagy is associated with the pathogenesis of a variety of diseases, including neurodegenerative diseases, myocardial diseases, inflammation, cancer, and aging.

[0003] Neurodegenerative Disorder refers to a disease caused by abnormal death of central neurons, which leads to dysfunction of the nervous system. To date, there is no fundamental treatment for neurodegenerative diseases that can slow down the progression of the disease. It is known that mitochondrial dysfunction is an important pathogenesis of Parkinson's disease (PD), Down syndrome (DS) and Alzheimer's disease (AD). DS is a neuronal developmental disease, but it is also associated with neurodegenerative diseases such as Alzheimer's disease. In neurons and brain organoids derived from DS patients, abnormal aggregation of defective mitochondria is often observed. In PD, mutations in PARKIN and PINK1, key regulators of mitochondria, prevent mitochondrial self-renewal, leading to mitochondrial dysfunction. In AD, the pathogenic protein Aβ1–42 interacts with the mitochondrial membrane and impairs mitochondrial function. Aβ1–42 also interferes with mitophagy regulators such as PINK1, thereby inhibiting mitophagy. In addition to neurodegenerative diseases, the decline of PARKIN and the mitochondrial receptor BNIP3 is associated with several types of cancer. Furthermore, the accumulation of damaged mitochondria can induce cGAS-STING-mediated inflammatory responses and inflammasome assembly-mediated cell death.

[0004] The accumulation of damaged mitochondria is known to induce cGAS-STING-mediated inflammatory responses and inflammasome assembly-mediated cell death.

[0005] Therefore, enhancing mitophagy is a promising approach to developing interventions for a variety of diseases. However, achieving this with small molecule compounds remains challenging. Several compounds, such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) of the phenylhydrazone class, and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) are known to induce mitophagy, but they damage mitochondria. Hirokazu Arimoto's group developed the autophagy targeting chimera (AUTAC) system. AUTAC can bind to target proteins, and part of the structure of AUTAC mimics S-guanylation modification and induces K63 polyubiquitination of target proteins. The ubiquitinated target proteins are then recognized and bound by the selective autophagy receptor p62 (SQSTM1 / p62), which further interacts with LC3 to mediate the recruitment of target proteins to autophagosomes. However, it is unclear how S-guanylation modification induces K63 ubiquitination, and the unclear mechanism makes the effects / side effects of AUTAC uncertain.

[0006] Urolithin A can improve Aβ pathology and cognitive decline in AD mouse models by enhancing mitochondrial autophagy. However, the molecular mechanism remains unclear. + Enhancers, such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), have strong effects on enhancing autophagy / mitochondrial autophagy, reducing cortical Aβ deposition and improving cognition in AD mouse models. Metformin can activate autophagy / mitochondrial autophagy through AMPK, thereby improving movement, learning, and memory in AD patients. Although some of the side effects or mechanisms of these compounds are unclear, their beneficial effects on AD mouse models or patients suggest that enhancing mitophagy is a viable strategy for diseases associated with defects in mitophagy or mitochondrial function.

[0007] There is an urgent need in the art for effective compounds that can be used to treat or prevent diseases associated with mitochondrial dysfunction. Summary of the invention

[0008] In one aspect, a compound of formula (I) is provided:

[0009]

[0010] or a pharmaceutically acceptable salt thereof,

[0011] in,

[0012] MTM is the mitochondrial binding part;

[0013] L is the joint part;

[0014] LCM is the LC3 binding moiety.

[0015] In one embodiment, the LCM moiety is a structure of the following formula (1) or a pharmaceutically acceptable salt thereof:

[0016]

[0017] in,

[0018] Y is O or S;

[0019] The C ring is selected from C 6-10 Aryl and five- to seven-membered heteroaryl, the aryl or heteroaryl is optionally substituted by one or more independently selected from R 1 The group substitution;

[0020] L 01 is a bond, or is a C1-C6 hydrocarbon chain;

[0021] R 01 , R 02 , R 03 , R 04 Each independently selected from H and R X2 ;

[0022] R X1 and R X2 is independently selected at each occurrence from halogen, -NO2, -CN, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR 05 、-SR 05 、-NR 05 R 06 、-C(=O)OR 05 、-C(=O)NR 05 R 06 、-OC(=O)R 05 、-NC(=O)R 05 R 06 、-C(=O)R 05 、-S(=O)2OR 05 、-S(=O)2R 05 、-S(=O)2NR 05 R 06 、-OS(=O)2R 05 、-NS(=O)2R 05 R 06 、-S(=O)R 05 wherein the alkyl, alkenyl or alkynyl is optionally substituted by one or more selected from halogen, -NO2, -CN, -OH, -O(C 1-6 Alkyl), -O(C 3-6 Cycloalkyl), -O(C 1-4 Alkylene-C3-6 Cycloalkyl), -O(three-membered to seven-membered heterocyclic group), -O(C 1-4 Alkylene)-(three-membered to seven-membered heterocyclic group), -SH, -S(C 1-6 Alkyl), -S(C 3-6 Cycloalkyl), -S(C 1-4 Alkylene-C 3-6 Cycloalkyl), -S(three-membered to seven-membered heterocyclic group), -S(C 1-4 Alkylene)-(three-membered to seven-membered heterocyclic group), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -NH(C 3-6 Cycloalkyl), -N(C 3-6 Cycloalkyl)2, -NH(C 1-4 Alkylene-C 3-6 Cycloalkyl), -N(C 1-4 Alkylene-C 3-6 Cycloalkyl)2, -NH(three-membered to seven-membered heterocyclic group), -N(three-membered to seven-membered heterocyclic group), -NH(C 1-4 Alkylene-three to seven-membered heterocyclic group), -N(C 1-4 alkylene-three to seven membered heterocyclic group)2, =O, -COOH and C 1-6 Substitution of alkyl groups;

[0023] R 05 , R 06 Each occurrence is independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl, three-membered to seven-membered heterocyclic group, three-membered to seven-membered heterocyclic group-C 1-4 Alkyl, C 6-10 Aryl-C 1-4 wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl is optionally substituted by one or more selected from halogen, -NO2, -CN, C 1-6 Alkyl, -OH, -O(C 1-6 Alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 alkyl)2, -COOH, -C(=O)O(C 1-6 alkyl), -C(=O)NH(C 1-6 alkyl), -C(=O)N(C 1-6 Alkyl)2, -OC(=O)(C 1-6 alkyl), -NHC(=O)(C1-6 alkyl), -C(=O)(C 1-6 alkyl) substituent.

[0024] In one embodiment, formula (1) has the structure of the following formula (2):

[0025]

[0026] in,

[0027] R 1 Selected from halogen, -NO2, -CN and C 1-6 Alkyl, wherein the alkyl is optionally substituted with one or more substituents selected from halogen, -NO2 and -CN;

[0028] R 2 , R 3 and R 4 is independently selected at each occurrence from halogen, -NO2, -CN, C 1-6 Alkyl, -OR 5 、-SR 5 、-NR 5 R 6 wherein the alkyl group is optionally substituted by one or more selected from halogen, -NO2, -CN, C 1-6 Alkyl, -OR 5 、-SR 5 、-NR 5 R 6 Substituents are substituted;

[0029] R 5 and R 6 is independently selected at each occurrence from hydrogen and C 1-6 alkyl.

[0030] In one aspect, compounds are provided:

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In the figures, C1 represents compound 1, C2 represents compound 2, L1 represents compound L1, and T1 represents compound T1. ns represents not significant. Figure 8 , 9 , 10, 11, 12, 14, 21-24, 25, 27-30, 37, 39, 40, 53, 55, 57, 59, 64-68, 7, 13, 1-3, 16-20, 32-36, 45-48 using t-test. Figure 4 , 5, 15, 26, 38, 6, 31a, 31b, 50a, 50b were analyzed using two-way ANOVA. Figure 8 , 10 , 11, 22, 23, 27, 28, 37, 39, 68, 7, 13, 2, 16-18, 32, 33, 36 Scale bar 20 μm. Fig. 9 , 21 , 25, 41a, 41b, 42-44, 52, 54, 58, 66 Scale bar: 10 μm. Fig.26 , 38 , 6, 3, 31a, 31b, 49a, 49b, 50a, 50bScale bar: 100 μm. Fig.56 Scale bar 500 μm.

[0033] Figure 1 : Compound 1 did not affect the expression of Flag-mito-keima.

[0034] Figure 2 : Effect of compound 1 on the red signal of Flag-mito-keima.

[0035] Figure 3 : Effects of compound 1, CQ or compound 1+CQ on the red signal of Flag-mito-keima.

[0036] Figure 4 : Effects of compound 1, compound 2, CCCP and compound T1 on mito-keima red signal.

[0037] Figure 5 : Effects of compound 1, CCCP and compound L1 on mito-keima red signal.

[0038] Figure 6 : Effect of compound 1 on mito-keima red signal.

[0039] Figure 7 : Effects of compound 1, CCCP, compound L1 and compound T1 on mito-keima red signal.

[0040] Figure 8 : Effects of compound 1 and CCCP on the red signal of mito-keima.

[0041] Fig. 9 : Effect of compound 1 on the colocalization of TOM20 and LAMP1.

[0042] Fig.10 : Effects of compound 1, compound T1 or compound L1 on mito-DsRed signal.

[0043] Fig.11 : Effects of compound 1 on TOM20 levels.

[0044] Fig.12 : Effects of compound 1 on TOM20 and UQCRC1 levels.

[0045] Fig.13 : Effects of compound 1 and CCCP on TOM20 levels.

[0046] Fig.14 : Effects of compound 1 on mtDNA levels.

[0047] Fig.15 : Effects of compound 1, CCCP or compound 1+CCCP on the red signal of mito-keima.

[0048] Fig.16 : Effect of compound 1 on TOM20 levels in HEK293-αsyn-A53T cells.

[0049] Fig.17 : Effects of compound 1 on TOM20 and UQCRC1 levels in HEK293-αsyn-A53T cells.

[0050] Fig.18 : Effects of compound 1, compound T1, compound L1 and CCCP on mitochondrial membrane potential.

[0051] Fig.19 : CCK8 assay showed that compound 1 did not cause cytotoxicity.

[0052] Fig. 20 : Compound 1 treatment did not change SQSTM1 / p62 or LC3B levels.

[0053] Fig.21 : Effects of compound 1, CQ, or compound 1+CQ on the colocalization of LC3 and mitochondria.

[0054] Fig. 22 : Effect of compound 1 on the percentage of mito-tracker positive area in WT and LC3B KO 293T cells.

[0055] Fig.23 , Fig.24 : Effects of compound 1 on TOM20 levels in WT and LC3B KO 293T cells.

[0056] Fig.25: Effects of compound 1, CQ, or compound 1+CQ on the colocalization of GFP-LC3B and TOM20 in WT or ATG5 KD HeLa cells.

[0057] Fig.26 : Effects of compound 1 on mito-keima red signal in WT or ATG5 KD HeLa cells.

[0058] Fig. 27 : Effects of compound 1 on TOM20 levels in WT or ATG5 KD HeLa cells.

[0059] Fig.28 : Effects of compound 1 on TOM20 levels in WT or ATG5 KO 293T cells.

[0060] Fig.29 : Effects of compound 1 on TOM20 and UQCRC1 levels in WT or ATG5 KO 293T cells.

[0061] Fig.30 : Compound 1 in WT or ATG9A KO STHdh Q7 / Q7 Effects on TOM20 levels in cells.

[0062] Fig.31a , 31b : Effects of compound 1, CQ, or compound 1+CQ on mito-keima red signal.

[0063] Fig.32 : Effects of compound 1, CQ or compound 1+CQ on the red signal of mito-DsRed.

[0064] Fig.33 , Fig.34 : Effects of compound 1, CQ or compound 1+CQ on TOM20 levels.

[0065] Fig.35 : Effects of compound 1, CQ or compound 1+CQ on mtDNA levels.

[0066] Fig.36 : Effects of compound 1, CQ, or compound 1+CQ on mito-QC signal.

[0067] Fig.37 : Effects of compound 1, CQ, or compound 1+CQ on the colocalization of mito-DsRed and GFP-LC3B in WT or TSPO KD HeLa cells.

[0068] Fig.38: Effects of compound 1 on mito-keima red signal in WT or TSPO KD HeLa cells.

[0069] Fig.39 , Fig.40 : Effects of compound 1 on TOM20 levels in WT or TSPO KD HeLa cells.

[0070] Fig.41a , 41b : Effect of compound 1 on the interaction between GST-LC3B and endogenous TSPO protein.

[0071] Fig.42 : Effect of compound 1 on the interaction between 6*His-TSPO and GST-LC3B.

[0072] Fig.43 : Effects of compound 1, CQ or compound 1+CQ on the interaction between GFP-LC3B and TSPO.

[0073] Fig.44 : Effects of compound 1, CQ or compound 1+CQ on the colocalization of GFP-LC3B and TSPO.

[0074] Fig.45 , Fig.46 : Effects of compound 1 on TOM20 levels in SH-SY5Y-αsyn-WT or SH-SY5Y-αsyn-A53T cells.

[0075] Fig.47 , Fig.48 :Compound 1 in MPP + Effects of induced cellular PD model on TOM20 levels.

[0076] Fig.49a , 49b and Fig.50a , 50b :Caspase-3 detection showed that compound 1 attenuated MPP in the cell PD model + Induced toxicity.

[0077] Fig.51 : iPSCs from DS patients were differentiated into cortical neurons and brain organoids and used for testing.

[0078] Fig.52 : Representative immunostaining images of cortical neurons in the euploid (IMR90-4) and triploid (2DS3) groups.

[0079] Fig.53: Compound 1 can alleviate the mitochondrial aggregation distribution in cortical neurons of the triploid (DS) group.

[0080] Fig.54 : Representative immunostaining images of brain organoids.

[0081] Fig.55 : Compound 1 can improve the aggregated mitochondria in DS brain organoids.

[0082] Fig.56 : Representative transmission electron microscopy (TEM) images of brain organoids from the euploid and triploid groups.

[0083] Fig.57 : Compound 1 can improve the mitochondrial aggregation distribution in brain organoids of euploid and triploid groups.

[0084] Fig.58 :Immunostaining detection of compound 1 on deep cortical neurons (CTIP2 + neurons).

[0085] Fig.59 :Compound 1 can increase CTIP2 in the triploid group + Neuron.

[0086] Fig.60 : Effects of compound 1 and CCCP, or AUTAC4 and CCCP on mito-tracker signal intensity in HEK293-αsyn-A53T cells or SH-SY5Y cells.

[0087] Fig.61 : AUTAC4 has little effect on mito-tracker signal intensity in HEK293-αsyn-A53T cells.

[0088] Fig.62 : AUTAC4 has little effect on TOM20 and UQCRC1 levels in HeLa cells.

[0089] Fig.63 : Effects of compound 1 and AUTAC4 on mito-keima red signal in HeLa cells.

[0090] Fig.64 : Methods by which the FKBP-FRB system promotes the interaction of LC3 with TSPO.

[0091] Fig.65 : The FKBP-FRB system promotes the interaction between LC3 and TSPO.

[0092] Fig.66: The interaction between LC3 and TSPO reduces the mito-tracker signal intensity and enhances the colocalization of FKBP-LC3B on mitochondria.

[0093] Fig.67 , Fig.68 : The interaction between LC3 and TSPO reduces TOM20 levels.

[0094] Fig.69 : The FKBP-FRB system promotes the interaction between PARKIN and TSPO.

[0095] Fig.70 :The FKBP-FRB system promotes the colocalization of GFP-PARKIN and mito-tracker.

[0096] Fig.71 : The interaction of PARKIN and TSPO cannot reduce TOM20 levels.

[0097] Fig.72 : The interaction of PARKIN with TSPO fails to increase the mito-keima red signal. DETAILED DESCRIPTION

[0098] Specific embodiments are provided below to illustrate the technical contents of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention through the contents disclosed in the specification. The present invention can also be implemented or applied through other different specific embodiments. Those skilled in the art can make various modifications and changes without departing from the spirit of the present invention.

[0099] definition

[0100] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. The technology used herein refers to the technology generally understood in the art, including variants and equivalent substitutions that are obvious to those skilled in the art. Although it is believed that the following terms are easily understood by those skilled in the art, the following definitions are set forth to better illustrate the present invention. When a trade name appears herein, it refers to the corresponding commodity or its active ingredient. All patents, published patent applications and publications cited herein are incorporated herein by reference.

[0101] When a certain amount, concentration or other numerical value or parameter is described in the form of a range, a preferred range or a preferred upper limit or a preferred lower limit, it should be understood as being equivalent to specifically disclosing any range formed by combining any upper limit or preferred value with any lower limit or preferred value, whether or not the range is explicitly stated. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range. For example, the expression "C1-C 10 ” or “C 1-10 " covers the range of 1-10 carbon atoms and should be understood to also cover any subranges and each point value therein, such as C 2-3 , C 2-4 , C 2-5 , C 3-4 , C 3-5 , C 3-6 , C 3-7 , C 1-2 , C 1-3 , C 1-4 , C 1-5 , C 1-6 , C 1-7 , C 1-8 , C1-9, etc., as well as C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 etc. The expression “C3-C 10 ” or “C 3-10 " should also be understood in a similar way, for example, it can cover C 3-4 , C 3-5 , C 3-6 , C 3-7 , C 3-8 , C 4-5 , C 4-6 , C4-C7, C5-C6, etc. and C3, C4, C5, C6, C7, C8, C9, C 10 For another example, the expression "3-7 yuan" should be understood to include any sub-ranges therein and each point value, such as 3-4, 3-5, 3-6, 3-7, 4-5, 4-6, 4-7, 5-6, 3, 4, 5, 6 or 7 yuan, etc.

[0102] When any variable (such as R L1 , R L2 , R L3 , R L4 , R L5 ) occurs more than once in the composition or structure of a compound, its definition is independent at each occurrence.

[0103] Unless the context clearly dictates otherwise, singular forms such as "a", "an", and "the" include plural forms. The expression "one or more" or "at least one" may mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. In one embodiment, "at least one" means 1, 2, 3 or 4.

[0104] The terms "optional" or "optionally" mean that the then described event may occur but does not necessarily occur, and that the description includes instances where said event or circumstance occurs or does not occur.

[0105] The terms "substituted" and "substituted" refer to one or more (e.g., one, two, three or four) hydrogen atoms on the designated atom being replaced by a selection from the indicated group, provided that the normal atomic valence of the designated atom in the current situation is not exceeded and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form stable compounds. When a substituent is described as not present, it should be understood that the substituent can be one or more hydrogen atoms, provided that the structure can achieve a stable state of the compound.

[0106] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any convenient position of the substituent. When a bond to a substituent is shown as passing through a bond connecting two atoms in a ring, then such a substituent may be bonded to any ring-forming atom in the substitutable ring.

[0107] The expressions "comprising," "including," "containing," and "having" are open ended and do not exclude additional unrecited elements, steps, or ingredients. The expression "consisting of excludes any element, step, or ingredient not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or ingredients, as well as the optional presence of elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" includes the expressions "consisting essentially of" and "consisting of.

[0108] The term "halo" or "halogen" or "halo" is understood to mean a fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atom, preferably a fluorine, chlorine or bromine atom.

[0109] The term "hydrocarbyl" refers to a monovalent group derived from a hydrocarbon. Examples of hydrocarbyl groups include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, and aryl.

[0110] The term "alkyl" refers to a saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. Alkyl groups include straight-chain alkyl groups and branched-chain alkyl groups. Alkyl groups can contain 1 to 10 carbon atoms, referred to as C 1-10 Alkyl groups, such as C1-6 Alkyl, C 1-4 Alkyl, C 1-3 Alkyl, C 1-2 Alkyl, C3 alkyl, C4 alkyl, C 3-6 Alkyl. Non-limiting examples of straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc. Non-limiting examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc.

[0111] A divalent group refers to a group obtained by removing a hydrogen atom from an atom with free valence electrons of a corresponding monovalent group. A divalent group has two attachment sites connected to the rest of the molecule, wherein the two attachment sites can be located on the same atom or two different atoms of the divalent group.

[0112] "Alkylene" or "alkylidene" refers to a saturated divalent hydrocarbon group. Alkylene includes straight chain or branched chain alkylene. Examples of straight chain alkylene include, but are not limited to, methylene (-CH2-), -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, etc. Examples of branched chain alkylene include, but are not limited to -CH(CH3)-, -CH(C2H5)-, -CH(CH3)-CH2-, -CH(C3H7)-, -CH(C2H5)-CH2-, -C(CH3)2-CH2-, -(CH(CH3))2-, -CH(CH3)-(CH2)2-, -CH2-CH(CH3)-CH2-, -CH(C4H9)-, -C(CH3)(C3H7)- , -C(C2H5)2-, -CH(C3H7)-CH2-, -CH(C2H5)-CH(CH3)-, -CH(C2H5)-(CH2)2-, -CH2-CH(C2H5)-CH2 -, -C(CH3)2-(CH2)2-, -CH2-C(CH3)2-CH2-, -CH(CH3)-(CH2)3-, -CH2-CH(CH3)-(CH2)2-, -CH(C5H 11)-, -C(C2H5)(C3H7)-, -C(CH3)(C4H9)-, -CH(C4H9)-CH2-, -C(C2H5)2-CH2-, -C (CH3)(C3H7)-CH2-, -CH(C2H5)-CH(C2H5)-, -CH(CH3)-CH(C3H7)-, -C(CH3)2-C( CH3)2-, -CH(C3H7)-(CH2)2-, -CH2-CH(C3H7)-CH2-, -CH(C2H5)-C(CH3)2-, -C(CH3)2-CH(CH3)-CH2-, -CH(CH3)-C(CH3)2-CH2-, -CH(C2H5)-CH(CH3)-CH2-, -CH( CH3)-CH(C2H5)-CH2-, -CH(CH3)-CH2-CH(C2H5)-, -CH(CH3)-C(CH3)2-CH2-, -(CH(CH3))3-, -C(CH3)2-(CH2)3-, -CH(C2H5)-(CH2)3-, -CH2-CH(C2H5)-(CH2)2-, -CH2-CH(CH3)-CH(CH3)-CH2-, -(CH(CH3))2-(CH2)2-, -CH(CH3)-(CH2)2-CH(CH3)-, -(CH2)2-CH(CH3)-(CH2)2-, -CH2-CH(CH3)-(CH2)3-, -CH(CH3)-(CH2)4-, etc.

[0113] The term "alkenyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond. The alkenyl group may have 2 to 8 carbon atoms, i.e., "C 2-8 Alkenyl", such as C 2-4 Alkenyl, C 3-4 Alkenyl. Non-limiting examples of alkenyl groups include, but are not limited to, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, and the like.

[0114] The term "alkynyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one triple bond. The alkynyl group may have 2 to 8 carbon atoms, i.e., "C 2-8 Alkynyl", such as C 2-4 Alkynyl, C 3-4 Alkynyl. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, and the like.

[0115] The term "cycloalkyl" refers to a saturated or unsaturated non-aromatic cyclic hydrocarbon group composed of carbon atoms and hydrogen atoms, preferably containing 1 or 2 rings. The cycloalkyl group can be a monocyclic, fused polycyclic, bridged or spirocyclic structure. The cycloalkyl group can have 3-10 carbon atoms, i.e., "C 3-10 Cycloalkyl", such as C 3-8 Cycloalkyl, C5 cycloalkyl, C6 cycloalkyl, C7 cycloalkyl. Non-limiting examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl and spiro[3.3]heptyl, etc. In some embodiments, the C atoms in the cycloalkyl are optionally substituted with oxo. In some embodiments, the C atoms in the cycloalkyl are optionally substituted with imino. In some embodiments, the imino is unsubstituted (=NH) or substituted with a group described in the context. In some embodiments, the imino is substituted with -OR, wherein R is H or C 1-10 alkyl.

[0116] The terms "cyclic alkyl" and "cycloalkyl" have the same meaning herein and can be used interchangeably. Cycloalkyl refers to a saturated cyclic hydrocarbon group. A cycloalkyl group can have 3, 4, 5, 6, 7, 8, 9 or 10 ring carbon atoms (C 3-10 ). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[1.1.1]pentyl, and bicyclo[2.1.1]hexyl. In some embodiments, the cycloalkyl group is C 3-7 Monocyclic or bicyclic cycloalkyl, preferably C 3-6 Monocyclic cycloalkyl, in particular cyclopropyl.

[0117] The term "heterocyclyl" or "heterocycloalkyl" refers to a monocyclic or bicyclic ring system (3-14-membered, 7-14-membered, 3-8-membered, 3-7-membered, 4-6-membered, 5-6-membered) having, for example, 3-14 ring atoms (e.g., 7-14, 3-8, 3-7, 4-6, or 5-6 ring atoms), wherein at least one ring atom (e.g., 1, 2, or 3) is a heteroatom selected from oxygen, sulfur, nitrogen, and phosphorus, and the remaining ring atoms are C. The ring system may be saturated (also understood as the corresponding "heterocycloalkyl") or unsaturated (i.e., having one or more double bonds and / or triple bonds in the ring). Optionally, the heterocyclyl may be benzo-fused. "Heterocyclyl" or "heterocycloalkyl" does not have aromaticity. In some embodiments, the C, N, S, and P atoms in the heterocycle are optionally substituted by oxo. In some embodiments, the C, S, and P atoms in the heterocycle are optionally substituted by imino. In some embodiments, the imino group is unsubstituted (=NH) or substituted with groups as described above or below. In some embodiments, the imino group is substituted with -OR, wherein R is H or C 1-10 alkyl.

[0118] The heterocyclic group can be, for example, a four-membered ring, such as azetidinyl, oxetanyl; or a five-membered ring, such as tetrahydrofuranyl, dioxolinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, oxopyrrolidinyl, 2-oxoimidazolidin-1-yl; or a six-membered ring, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, 1,1-dioxo-1,2-thiazin-2-yl or trithianyl; or a seven-membered ring, such as diazepine, Base ring.

[0119] The heterocyclic group may be bicyclic, without limitation, for example, a five-membered and a five-membered ring, such as octahydrocyclopenta[c]pyrrolyl; or a five-membered and a six-membered bicyclic ring, such as octahydropyrrolo[1,2-b]pyrazinyl.

[0120] As mentioned above, the heterocycle may be unsaturated, i.e., it may contain one or more double bonds, without being limited thereto, for example, an unsaturated heterocycle containing a nitrogen atom may be a 1,6-dihydropyrimidine, a 1,2-dihydropyrimidine, a 1,4-dihydropyrimidine, a 1,6-dihydropyridine, a 1,2-dihydropyridine, a 1,4-dihydropyridine, a 2,3-dihydro-1H-pyrrole, a 3,4-dihydro-1H-pyrrole, a 2,5-dihydro-1H-pyrrolyl, a 4H-[1,3,4]thiadiazinyl, a 4,5-dihydrooxazolyl or a 4H-[1,4]thiazinyl ring, an unsaturated heterocycle containing an oxygen atom may be a 2H-pyran, a 4H-pyran, a 2,3-dihydrofuran, an unsaturated heterocycle containing a sulfur atom may be a 2H-thiopyran, a 4H-thiopyran. The heterocycle may be benzo-fused, without being limited thereto, for example, a dihydroisoquinolinyl ring.

[0121] Exemplary bicyclic heterocycles also include:

[0122]

[0123] The term "aryl" refers to an aromatic ring group of an all-carbon monocyclic or fused polycyclic (such as a bicyclic) ring having a conjugated π electron system. For example, an aryl group may have 6-14 carbon atoms, preferably 6-10, more preferably 6 or 10. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, and the like.

[0124] The term "heteroaryl" is to be understood as preferably meaning a monovalent monocyclic, bicyclic or tricyclic aromatic ring system having 5, 6, 7, 8, 9 or 10 ring atoms ("5-10 heteroaryl"), in particular 5 or 6 or 9 or 10 ring atoms, and containing at least one (suitably 1 to 4, more suitably 1, 2 or 3) heteroatom which may be identical or different, such as oxygen, nitrogen or sulfur. Furthermore, in each case the heteroaryl group may be benzo-fused. In particular, the heteroaryl group is selected from thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, and the like, and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl, and the like; or azocinyl, indolizinyl, purinyl, and the like, and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, carbazolyl, acridinyl, and the like.

[0125] The term "hydrocarbon chain" refers to a chain group consisting of carbon atoms and hydrogen atoms, which may be straight or branched. The hydrocarbon chain may be saturated (i.e., alkylene) or unsaturated, i.e., may contain one or more (preferably 1) carbon-carbon double bonds or triple bonds. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2-), 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), 1,7-heptylene (-CH2CH2CH2CH2CH2CH2CH2-), and the like.

[0126] When depicting a chemical bond connected to a molecule or a part of a molecule herein, if the specific atom connected by the chemical bond is not depicted, it means that the chemical bond can connect any atom of the molecule or the part, as long as a stable structure can be formed. For example, a part of a molecule can be depicted and placed in brackets, such as parentheses or square brackets; when depicting other parts of the molecule connected to the part in the brackets by a chemical bond, if the specific atom in the brackets connected by the chemical bond is not depicted, it means that the chemical bond can connect any atom of the part in the brackets, as long as a stable structure can be formed.

[0127] The pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof. The methods for preparing the pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0128] The compounds of the present invention encompass pharmaceutically acceptable salts, stereoisomers, solvates, polymorphs, tautomers, isotopic compounds, metabolites or prodrugs thereof.

[0129] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds. For example, the compounds of the present invention may exist in E or Z configurations of carbon-carbon double bonds or carbon-nitrogen double bonds. The compounds of the present invention may also exist in the form of a mixture of "E" and "Z" isomers. Isomer forms also include cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Purification and separation of such substances can be achieved by standard techniques known in the art.

[0130] Optically pure enantiomers can be obtained by resolving racemic mixtures according to conventional methods, for example, by forming diastereomeric salts using optically active acids or bases, or by forming covalent diastereomers. A mixture of diastereoisomers can be separated into single diastereomers based on their physical and / or chemical differences by methods known in the art (e.g., by chromatography or fractional crystallization). Then, the optically active enantiomer base or acid is released from the separated diastereomeric salts. Another method for separating racemic enantiomers can use chiral chromatography (e.g., chiral HPLC columns), and the separated chiral isomers can be subjected to conventional derivatization treatment or non-derivatization before separation, depending on which method can achieve more effective separation of chiral isomers. Enzymatic methods can also be used to separate derivatized or non-derivatized chiral isomers. Similarly, optically active raw materials can be used to obtain optically pure compounds of the present invention by chiral synthesis.

[0131] The compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain a solvent as a structural element of the crystal lattice of the compound, in particular water, methanol or ethanol. The amount of polar solvents, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

[0132] The present invention also encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be a single polymorph or a mixture of more than one polymorph in any ratio.

[0133] The compounds of the invention may exist in isotopically labeled or enriched form, containing one or more atoms having a different mass and mass number from the most common atomic mass in nature. Isotopes may be radioactive or non-radioactive isotopes. Isotopes of atoms such as hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine include, but are not limited to, 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 32 P. 35 S. 18 F. 36 Cl and 125 I.

[0134] Also included within the scope of the present invention are metabolites of the compounds of the present invention, i.e., substances formed in vivo when the compounds of the present invention are administered. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymolysis, etc. of the administered compounds.

[0135] The present invention further includes within its scope prodrugs of the compounds of the invention, which are certain derivatives of the compounds of the invention that may themselves have little or no pharmacological activity but are converted, for example by hydrolytic cleavage, into compounds of the invention having the desired activity when administered into or onto the body.

[0136] The term "polymorph" or "polymorph" refers to a single polymorph or a mixture of more than one polymorph in any proportion.

[0137] The term "crystalline form" or "crystal" refers to any solid material that exhibits a three-dimensional ordering, in contrast to amorphous solid material, which produces a characteristic X-ray powder diffraction pattern with well-defined peaks.

[0138] The term "amorphous" refers to any solid material that has no order in three dimensions.

[0139] The term "pharmaceutically acceptable" means that it is within the scope of normal medical judgment and will not cause undue toxicity, irritation, allergic response, or the like in contact with the tissues of patients.

[0140] The term "pharmaceutically acceptable carrier" refers to those substances that have no significant irritation to organisms and do not impair the biological activity and performance of the active compound. "Pharmaceutically acceptable carrier" includes, but is not limited to, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, disintegrants, stabilizers, solvents or emulsifiers.

[0141] The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating or preventing a target disorder, disease, or condition.

[0142] With respect to a drug, drug unit or active ingredient, the term "effective amount", "therapeutically effective amount" or "prophylactically effective amount" refers to a sufficient amount of the drug or pharmaceutical agent that can achieve the desired effect with acceptable side effects. The determination of the effective amount varies from person to person, depending on the age and general condition of the individual and on the specific active substance. The appropriate effective amount in each case can be determined by a person skilled in the art based on routine experiments.

[0143] The term "subject" includes humans or non-human animals. Exemplary human subjects include human subjects (referred to as patients) suffering from diseases (e.g., diseases described herein) or normal subjects. "Non-human animals" in the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0144] Compounds of formula (I)

[0145] In one aspect, the present invention provides a compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof:

[0146]

[0147] in,

[0148] MTM is the mitochondrial binding part;

[0149] L is the joint part;

[0150] LCM is the LC3 binding moiety.

[0151] In one embodiment, the LC3 binding moiety refers to a moiety that has affinity for the LC3 protein.

[0152] In one embodiment, the LCM moiety is a structure of the following formula (1) or a pharmaceutically acceptable salt thereof:

[0153]

[0154] in,

[0155] Y is O or S;

[0156] The C ring is selected from C 6-10 Aryl and five- to seven-membered heteroaryl, the aryl or heteroaryl is optionally substituted by one or more independently selected from R 1 The group substitution;

[0157] L01 is a bond, or is a C1-C6 hydrocarbon chain;

[0158] R 01 , R 02 , R 03 , R 04 Each independently selected from H and R X2 ;

[0159] R X1 and R X2 is independently selected at each occurrence from halogen, -NO2, -CN, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR 05 、-SR 05 、-NR 05 R 06 、-C(=O)OR 05 、-C(=O)NR 05 R 06 、-OC(=O)R 05 、-NC(=O)R 05 R 06 、-C(=O)R 05 、-S(=O)2OR 05 、-S(=O)2R 05 、-S(=O)2NR 05 R 06 、-OS(=O)2R 05 、-NS(=O)2R 05 R 06 、-S(=O)R 05 wherein the alkyl, alkenyl or alkynyl is optionally substituted by one or more selected from halogen, -NO2, -CN, -OH, -O(C 1-6 Alkyl), -O(C 3-6 Cycloalkyl), -O(C 1-4 Alkylene-C 3-6 Cycloalkyl), -O(three-membered to seven-membered heterocyclic group), -O(C 1-4 Alkylene)-(three-membered to seven-membered heterocyclic group), -SH, -S(C 1-6 Alkyl), -S(C 3-6 Cycloalkyl), -S(C 1-4 Alkylene-C 3-6 Cycloalkyl), -S(three-membered to seven-membered heterocyclic group), -S(C 1-4 Alkylene)-(three-membered to seven-membered heterocyclic group), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -NH(C 3-6Cycloalkyl), -N(C 3-6 Cycloalkyl)2, -NH(C 1-4 Alkylene-C 3-6 Cycloalkyl), -N(C 1-4 Alkylene-C 3-6 Cycloalkyl)2, -NH(three-membered to seven-membered heterocyclic group), -N(three-membered to seven-membered heterocyclic group), -NH(C 1-4 Alkylene-three to seven-membered heterocyclic group), -N(C 1-4 alkylene-three to seven membered heterocyclic group)2, =O, -COOH and C 1-6 Substitution of alkyl groups;

[0160] R 05 , R 06 Each occurrence is independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl, three-membered to seven-membered heterocyclic group, three-membered to seven-membered heterocyclic group-C 1-4 Alkyl, C 6-10 Aryl-C 1-4 wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl is optionally substituted by one or more selected from halogen, -NO2, -CN, C 1-6 Alkyl, -OH, -O(C 1-6 Alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 alkyl)2, -COOH, -C(=O)O(C 1-6 alkyl), -C(=O)NH(C 1-6 alkyl), -C(=O)N(C 1-6 Alkyl)2, -OC(=O)(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -C(=O)(C 1-6 alkyl) substituent.

[0161] In one embodiment, Y is O. When Y is O, formula (1) is In one embodiment, formula (1) is

[0162] In a preferred embodiment, the C ring is a benzene ring or a five-membered heteroaryl group, wherein the heteroaryl group is optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R X1In one embodiment, the five-membered heteroaryl contains 1, 2, 3 or 4 heteroatoms, each of which is independently selected from N, O and S, preferably N and O. In another embodiment, the five-membered heteroaryl contains at least one N atom. In one embodiment, the C ring is a five-membered heteroaryl containing 1 or 2 N atoms, optionally substituted by 1, 2, 3, 4 or 5 each independently selected from R X1 In one embodiment, the C ring is a benzene ring. In another embodiment, the C ring is a pyrrole.

[0163] In one embodiment, L 01 is a C1-C2 hydrocarbon chain. 01 In a particular embodiment, L 01 In another particular embodiment, L 01 for Wherein "" indicates the site where the structure is connected to ring C, Represents another point at which the structure is attached to the rest of the molecule.

[0164] In one embodiment, the structure of formula (1) is selected from the following formula (1-i), (1-ii) and formula (1-iii):

[0165]

[0166] In one embodiment, the structure of formula (1) is selected from the following formula (1-1), (1-2) and formula (1-3):

[0167]

[0168] In one embodiment, formula (1) has the structure of the following formula (2):

[0169]

[0170] in,

[0171] R 1 Selected from halogen, -NO2, -CN and C 1-6 Alkyl, wherein the alkyl is optionally substituted with one or more substituents selected from halogen, -NO2 and -CN;

[0172] R 2 , R 3 and R 4 is independently selected at each occurrence from halogen, -NO2, -CN, C 1-6 Alkyl, -OR 5 、-SR 5 、-NR 5 R6 wherein the alkyl group is optionally substituted by one or more selected from halogen, -NO2, -CN, C 1-6 Alkyl, -OR 5 、-SR 5 、-NR 5 R 6 Substituents are substituted;

[0173] R 5 and R 6 is independently selected at each occurrence from hydrogen and C 1-6 alkyl.

[0174] In one embodiment, R 1 is selected from halogen, in particular I or Br.

[0175] In one embodiment, R 2 is selected from halogen, especially Br.

[0176] In one embodiment, R 3 Selected from halogen and -OR 5 , especially -OR 5 In one embodiment, R 3 For OH.

[0177] In one embodiment, R 4 Selected from halogen and -OR 5 In one embodiment, R 4 is Br. In one embodiment, R 4 For OH.

[0178] In one embodiment, formula (2) is

[0179] In a particular embodiment, the structure of formula (2) is selected from the following formula (2-i) and formula (2-ii):

[0180]

[0181] In a particular embodiment, the structure of formula (2) is selected from the following formula (2-1) and formula (2-2):

[0182]

[0183] In a particular embodiment, the above formula (1-1) and formula (2-1) correspond to compound 1 of WO2020182143A1; formula (1-2) and formula (2-2) correspond to compound 3 of WO2020182143A1; formula (1-3) corresponds to compound 5 of WO2020182143A1; formula (1-4) corresponds to compound 6 of WO2020182143A1. WO2020182143A1 discloses the affinity activity of these compounds with LC3B.

[0184] L is a chemical bond or group for connecting the LCM part and the MTM part. In one embodiment, L is rigid or flexible. In a preferred embodiment, L is flexible. In one embodiment, L is a chemical bond. In another embodiment, L is a straight or branched hydrocarbon chain comprising 1-60, preferably 1-30, more preferably 2-16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16) carbon atoms, each of which may be optionally replaced by one or more, e.g., 1-3, preferably 1-2, especially 1 heteroatom; wherein the heteroatom is selected from oxygen, sulfur, nitrogen, phosphorus, preferably oxygen, sulfur or nitrogen, more preferably oxygen or nitrogen, especially oxygen; wherein the carbon atom or heteroatom is optionally replaced by one or more selected from R L1 , R L2 , R L3 , R L4 and R L5 substituted by a group; wherein

[0185] Each R L1 , R L2 , R L3 , R L4 and R L5 Each independently selected from: H, halogen, C 1-6 Alkyl, -O(C 1-6 Alkyl), -S(C 1-6 Alkyl), -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, C 3-6 Cycloalkyl, C 6-10 Aryl, five-membered to seven-membered heteroaryl, three-membered to seven-membered heterocyclic group, -O(C 3-6 Cycloalkyl), -S(C 3-6 Cycloalkyl), -NH(C 3-6 Cycloalkyl), -N(C 3-6 Cycloalkyl)2, -N(C 3-6 Cycloalkyl)(C 1-6 alkyl), -OH, -NH2, -SH, -S(=O)2(C 1-6 alkyl), -P(=O)(OC1-6 Alkyl)(C 1-6 alkyl), -P(=O)(OC 1-6 Alkyl)2, -C≡CC 1-6 Alkyl, -C≡CH, -CH=CH(C 1-6 Alkyl), -C(C 1-6 alkyl)=CH(C 1-6 Alkyl), -C(C 1-6 alkyl)=C(C 1-6 Alkyl)2, -Si(OH)3, -Si(C 1-6 Alkyl)3, -Si(OH)(C 1-6 Alkyl)2, -C(=O)(C 1-6 alkyl), -COOH, halogen, -CN, -CF3, -CHF2, -CH2F, -NO2, -S(=O)2NH(C 1-6 alkyl), -S(=O)2N(C 1-6 Alkyl)2, -S(=O)NH(C 1-6 alkyl), -S(=O)N(C 1-6 alkyl)2, -C(=O)NH(C 1-6 alkyl), -C(=O)N(C 1-6 Alkyl)2, -N(C 1-6 alkyl)C(=O)NH(C 1-6 Alkyl), -N(C 1-6 alkyl)C(=O)N(C 1-6 Alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH2, -N(C 1-6 Alkyl)S(=O)2NH(C 1-6 Alkyl), -N(C 1-6 alkyl)S(=O)2N(C 1-6 Alkyl)2, -NHS(=O)2NH(C 1-6 alkyl), -NHS(=O)2N(C 1-6 The heteroatom may be located in the middle of the chain or at one or both ends of the chain. When the heteroatom is located at the end, it means that the linker is connected to the LCM part and / or the MTM part through the heteroatom. In one embodiment, L is a straight chain group.

[0186] In another embodiment, L is a structure of formula (i) or a pharmaceutically acceptable salt thereof:

[0187] -Dy-(i)

[0188] Wherein, y is an integer greater than 1; and

[0189] Each D is independently selected from the group consisting of: a bond, -CR L1 R L2 -, -O-, -S-, -S(=O)-, S(=O)2-, -NR L3 -、-S(=O)2NR L3 -、-S(=O)NR L3 -、-C(=O)NR L3 -、-NR L3 C(=O)NR L4 -、-NR L3 S(=O)2NR L4 -, -C(=O)-, -CR L1 =CR L2 -、-C≡C-、-SiR L1 R L2 -、-P(=O)R L1 -、-P(=O)OR L1 -, optionally through 0-6 R L1 and / or R L2 C 3-6 Cycloalkyl, optionally with 0-6 R L1 and / or R L2 A three- to seven-membered heterocyclic group substituted by a group, optionally substituted by 0-6 R L1 and / or R L2 The aryl group substituted by a group, optionally substituted by 0-6 R L1 and / or R L2 A heteroaryl group substituted with a group; wherein

[0190] Each R L1 , R L2 , R L3 , R L4 and R L5 Each independently selected from: H, halogen, C 1-6 Alkyl, -O(C 1-6 Alkyl), -S(C 1-6 Alkyl), -NH(C 1-6 Alkyl), -N(C 1-6 alkyl)2, -OH, -NH2, -SH, halogen, -CN, -CF3, -CHF2, -CH2F and -NO2;

[0191] When y is greater than 1, R L1 or R L2 Each independently can be linked to another D group to form a further R L5The cycloalkyl and / or heterocyclyl moieties are substituted by radicals.

[0192] In one embodiment, each R L1 , R L2 , R L3 , R L4 and R L5 are each independently selected from H, halogen, C 1-6 Alkyl, -OH, -NH2, -SH, halogen, -CN, -CF3, -CHF2, -CH2F and -NO2.

[0193] In a particular embodiment, y is 14, and D is selected from -CR L1 R L2 -、-O-、-NR L3 -、-C(=O)NR L3 - and -C(=O)-. In one embodiment, R L1 , R L2 and R L3 In another specific embodiment, the structure of L is

[0194] In one embodiment, the mitochondrial binding moiety is a moiety that is capable of non-covalently interacting with mitochondria. In another embodiment, the MTM moiety is selected from molecules that have affinity for mitochondria. In one embodiment, the MTM moiety is capable of non-covalently interacting with mitochondrial outer membrane proteins (OMM). In one embodiment, the OMM is selected from mitochondrial outer membrane transport proteins (e.g., TOM20, TOM40, TOM70, TSPO), Synaptojanin-2 binding proteins (e.g., OMP25), SAM50. In a particular embodiment, the OMM is TSPO. In one embodiment, the MTM moiety is selected from molecules that have affinity for TSPO, preferably 2-phenyl-1H-indole. In one embodiment, the mitochondrial binding moiety is

[0195] In one embodiment, formula (I) has the structure of formula (II-i), wherein the LCM moiety is the structure of formula (1) above (see formula (1) and its embodiments above) or a pharmaceutically acceptable salt thereof

[0196]

[0197] Among them, Y, L 01 , R 01 , R 02 , R 03 , R 04 As defined in formula (1).

[0198] In one embodiment, L may be covalently linked to any suitable position of formula (1).

[0199] In one embodiment, formula (I) has the structure of formula (II-1):

[0200]

[0201] Among them, Y, L 01 , R 01 , R 02 , R 03 , R 04 As defined in formula (1).

[0202] In one embodiment, the compound of formula (II-1) is selected from the following compounds i1, i3 and i4, in particular compound i1:

[0203]

[0204] In one embodiment, the compound of formula (II-1) is selected from the following compound 1, compound 3 and compound 4, in particular compound 1:

[0205]

[0206] In one embodiment, formula (I) has the structure of formula (II-ii):

[0207]

[0208] Among them, Y, L 01 , R 01 , R 02 , R 03 , R 04 As defined in formula (1).

[0209] In one embodiment, formula (I) has the structure of formula (II-2):

[0210]

[0211] Among them, Y, L 01 , R 01 , R 02 , R 03 , R 04 As defined in formula (1).

[0212] In one embodiment, the compound of formula (II-2) is compound il, compound i3 and compound i4 as described above. In one embodiment, the compound of formula (II-2) is compound 1, compound 3 or compound 4 as described above.

[0213] Pharmaceutical compositions and pharmaceutical preparations

[0214] Another object of the present invention is to provide a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof, and at least one pharmaceutically acceptable carrier.

[0215] The pharmaceutical composition of the present invention can be administered in any manner as long as it achieves the effect of preventing, alleviating, preventing or treating human or animal symptoms. For example, various suitable dosage forms can be prepared according to the route of administration. For example, it can be administered orally or parenterally to a patient in the form of a conventional preparation. The conventional preparations are such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions and emulsions.

[0216] The dosage of the compound administered to a subject can be adjusted to a considerable extent. The dosage can vary depending on the specific route of administration and the needs of the subject, and can be subject to the judgment of a healthcare professional.

[0217] Treatments and uses

[0218] According to certain embodiments of the present invention, mitochondria can be targeted to lysosomes using the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotopic compound, metabolite or prodrug thereof, or a pharmaceutical composition of the present invention.

[0219] Therefore, in yet another aspect, the present invention also provides use of the compound of the present invention or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present invention for targeting mitochondria to lysosomes.

[0220] In another aspect, the present invention provides a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention, for use in targeting mitochondria to lysosomes.

[0221] In a further aspect, the present invention provides a method for targeting mitochondria to lysosomes, the method comprising administering to a subject in need thereof an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention.

[0222] In a preferred embodiment, the mitochondria targeted to the lysosome are damaged or dysfunctional mitochondria. The damage includes but is not limited to depolarization damage. In a preferred embodiment, the targeting refers to translocation of mitochondria into lysosomes.

[0223] According to certain embodiments of the present invention, the present invention or its pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug or the pharmaceutical composition of the present invention can be used to promote mitochondrial degradation.

[0224] Therefore, in another aspect, the present invention also provides use of the compound of the present invention or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present invention for promoting mitochondrial degradation.

[0225] In another aspect, the present invention provides a compound of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the present invention for use in promoting mitochondrial degradation.

[0226] In a further aspect, the present invention provides a method for promoting mitochondrial degradation, comprising administering an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention to a subject in need thereof.

[0227] In a preferred embodiment, the degraded mitochondria are damaged or dysfunctional mitochondria. The damage includes but is not limited to depolarization damage.

[0228] According to certain embodiments of the present invention, the present invention or its pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug or the pharmaceutical composition of the present invention can be used to prevent or treat diseases associated with mitochondrial dysfunction, such as neurodegenerative diseases, myocardial diseases, inflammation, cancer and aging.

[0229] Therefore, in another aspect, the present invention also provides the use of a compound of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease, disorder or condition selected from a disease associated with mitochondrial dysfunction, wherein the compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition is optionally used in combination with a second therapeutic agent.

[0230] In another aspect, the present invention provides a compound of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the present invention, optionally in combination with a second therapeutic agent, for use in treating a disease associated with mitochondrial dysfunction.

[0231] In a further aspect, the present invention provides a method for treating a disease associated with mitochondrial dysfunction, the method comprising administering to an individual in need thereof an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention, wherein the compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition is optionally used in combination with a second therapeutic agent.

[0232] In one embodiment, the disease, disorder or condition is a neurodegenerative disease, for example, selected from Parkinson's disease (PD), Down syndrome (DS) and Alzheimer's disease (AD). In a specific embodiment, the disease, disorder or condition is a neurodegenerative disease, for example, selected from Parkinson's disease (PD) and Down syndrome (DS).

[0233] In one embodiment, the disease, disorder or condition is a myocardial disease, eg, selected from heart failure and dilated cardiomyopathy (DCM).

[0234] In one embodiment, the disease, disorder or condition is selected from acute and chronic inflammatory diseases such as arthritis, inflammatory bowel disease, tendinitis, hepatitis, pancreatitis, vasculitis, contact dermatitis, psoriatic arthritis, ankylosing spondylitis and systemic lupus erythematosus (lupus).

[0235] In one embodiment, the disease, disorder or condition is cancer, for example selected from: (a) a solid tumor or a blood-borne tumor selected from the following cancers: bladder cancer, endometrial cancer, squamous cell lung cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, esophageal cancer, gallbladder cancer, brain cancer, head and neck cancer, ovarian cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer, prostate cancer and skin cancer; (b) a hematopoietic tumor of the lymphoid lineage selected from the following: leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's (c) a hematopoietic neoplasm of the myeloid lineage selected from the group consisting of acute and chronic myeloid leukemias, myelodysplastic syndromes and promyelocytic leukemias; (d) a tumor of mesenchymal origin selected from the group consisting of fibrosarcoma and rhabdomyosarcoma; (e) a tumor of the central and peripheral nervous system selected from the group consisting of astrocytoma, neuroblastoma, glioma and schwannoma; or (f) a melanoma, a seminoma, a teratoma, an osteosarcoma, a xeroderma pigmentosum, a keratoacanthoma, a follicular thyroid carcinoma or a Kaposi's sarcoma.

[0236] In one embodiment, the disease, disorder or condition is aging.

[0237] Combination therapy

[0238] The compound of the present invention or its pharmaceutically acceptable salt or the pharmaceutical composition of the present invention can be used alone or in combination with other therapeutic agents.

[0239] For example, the use of an adjuvant can enhance the therapeutic effect of the compounds of the present invention (for example, the therapeutic benefit of an adjuvant alone is minimal, but when used in combination with another drug, the therapeutic benefit of an individual can be enhanced), or, for example, the combination of the compounds of the present invention with another therapeutic agent that also has therapeutic effects can enhance the therapeutic benefit of an individual. For example, when treating cancer, when using the compounds of the present invention, the combined use of another therapeutic drug may enhance the clinical benefit. Therapies that can be combined include, but are not limited to, physical therapy, psychotherapy, radiotherapy, small molecule therapeutic agents, and the like. Regardless of the disease, disorder, or condition, the two therapies should have an additive or synergistic effect to benefit the individual's treatment.

[0240] Beneficial Effects

[0241] The present invention demonstrates for the first time that a bifunctional compound that binds to the mitochondrial outer membrane protein TSPO and the autophagosome protein LC3B can simultaneously enhance the phagocytosis of mitochondria by autophagosomes and the subsequent autophagic degradation.

[0242] The compounds of the present invention can selectively promote mitochondrial degradation by targeting autophagy, and preferentially degrade damaged mitochondria. And have no effect on the autophagic function and overall autophagic level of cells. Therefore, the compounds of the present invention can be effectively used to prevent and treat diseases related to mitochondrial dysfunction, and have good safety.

[0243] Example

[0244] In order to more clearly illustrate the purpose and technical scheme of the present invention, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The specific experimental methods not mentioned in the following examples are all carried out according to conventional experimental methods.

[0245] Experimental materials, reagents and procedures

[0246] Unless otherwise specified, the instruments and reagents used in the examples are commercially available.

[0247] Data were analyzed using GraphPad Prism 6 (GraphPad software, San Diego, USA). All data are expressed as mean ± standard deviation (SEM). Unless otherwise stated, statistical significance was tested using a two-tailed unpaired t-test or a two-way analysis of variance with Bonferroni's post hoc test. In all statistical analyses in this article, * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001, and **** indicates p < 0.0001.

[0248] Compound

[0249] Compound T1: 2-phenyl-1H-indole, PubChem CID: 13698.

[0250] Compound L1: GW5074, available from DC Chemicals, catalog number DC8810.

[0251] 5,7-Dihydroxy-4-phenyl-2H-coumarin: Also known as DP. PubChem CID 5398649, can be purchased from ChemDiv, catalog number D715-2435.

[0252] Other compounds such as m-chlorophenylhydrazone carbonyl cyanide (CCCP, CAS No.: 555-60-2) can be purchased from Merck and other companies.

[0253] Preparation of compounds

[0254] 1 HNMR was measured using a Bruker AVANCE III 400 MHz NMR spectrometer with tetramethylsilane (TMS) as the internal standard. The chemical shift was expressed in 10 -6 (ppm) is given as the unit.

[0255] The abbreviations in the nuclear magnetic resonance (NMR) data used in the preparation examples are shown below:

[0256] s: singlet, d: doublet, t: triplet, q: quartet, dd: double of doublet, qd: quadruple of doublets, ddd: double of doubles, ddt: double of doubles triplet, dddd: double of doubles of doublets, m: multiplet, br: broad, J: coupling constant, Hz: Hertz.

[0257] Mass spectrometer model: Agilent 1100LC / 1946D.

[0258] Unless otherwise specified, the reaction temperature is room temperature (15°C to 25°C).

[0259] Unless otherwise stated, the starting materials used in the preparation examples were purchased from commercial suppliers and used without further purification.

[0260] Cell culture

[0261] HeLa, 293T and HEK293-αsyn-A53T cells were cultured in DMEM supplemented with 10% FBS (Gibco, Waltham, USA, 10099-141). SH-SY5Y cells were cultured in DMEM / F-12 (Gibco, 11330-032) supplemented with 10% FBS, 1 mM sodium pyruvate, NEAA (Gibco, 11140050) in a cell culture incubator cultured at 37°C with a mixed humidified atmosphere of 5% CO2 and 95% air.

[0262] The 21 trisomy hPSC cell line (2DS3) was prepared according to the literature method (Tang et al., "DSCAM / PAK1pathway suppression reverses neurogenesis deficits in iPSC-derived cerebralorganoids from patients with Down syndrome." The Journal of clinical investigation, 2021, 131(12): e135763.; Weick et al., "Deficits in human trisomy21iPSCs and neurons." Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(24), pp 9962-9967). The euploid hPSC cell line (IMR90-4) was obtained from WiCell (Agreement NO.16-W0063). The hPSC was maintained on a vitronectin-coated cell culture plate using a nutrient-free culture technique. hPSCs were cultured in Essential 8 (E8) medium (Life Technologies, A15169-01) at 37°C in a cell culture incubator with a humidified atmosphere of 5% CO2 and 95% air. After 5-7 days of incubation, hPSCs were dissociated with EDTA (Lonza, Basel, Switzerland, 17-711E) at 37°C for 1-2 minutes and then incubated with 1×10 5 The cells were seeded into multi-well plates at a density of 10 cells / well for subculturing.

[0263] For experiments where images were captured using a confocal microscope, cells were seeded onto coverslips (Marienfeld, Lauda- Germany, 0111520) in multi-well plates overnight, and then proceed with subsequent processing (e.g. transfection, immunofluorescence staining).

[0264] Transfection

[0265] Using PolyJet TM The plasmid was transfected into cells using the ELISA kit (SignaGen, Jinan, China, SL100688). Subsequent treatment (eg, treatment with compounds) was performed 24-48 hours after transfection.

[0266] siRNA knockdown

[0267] siRNA transfection reagent: Lipofectamine 2000 (Life Technologies, Carlsbad, USA, 11668).

[0268] siRNA targeting ATG5 was purchased from Jima Pharmaceutical Technology Co., Ltd. (Shanghai, China).

[0269] The siRNA sequences are shown in the following table:

[0270]

[0271] The interference efficiency was assessed by Western blotting.

[0272] Plasmids

[0273] FKBP-LC3B, TSPO-Flag-FRB, Flag-TSPO, Flag-mito-keima and FKBP-GFP-Parkin plasmids were prepared by Sangon Biotech (Shanghai) Co., Ltd. The target sequence was subcloned into the pCDNA3.1(+) vector through HindIII and BamHI restriction enzyme sites. EGFP-LC3B (11546) and LAMP1-mcherry (45147) were purchased from Addgene (Watertown, USA). Mito-DsRed plasmid was purchased from Clontech Laboratories Inc (Beijing, China, 632421). Mito-QC (mito-mCherry-GFP) was from Sun Qiming of Zhejiang University. Mito-keima was from Xia Hongguang of Zhejiang University.

[0274] Differentiation of hPSCs into brain organoids

[0275] hPSC is cultured in a culture dish for 5-7 days to form clones of appropriate size with smooth edges. Differentiation is operated when the cell density is about 70%. hPSC is incubated in dispase (1U / mL, Gibco, 17105-041) for 2 minutes to separate and form embryoid bodies (EBs). EBs are then suspended in neural induction medium (NIM: 98% F12+1% NEAA+1% N2) for 7 days. On the 7th day, EBs are incubated with 10% fetal bovine serum (FBS) in neural induction medium for 8-10 hours to attach to multi-well plates. The culture medium is replaced with neural induction medium, and then the neural induction medium is replaced every two days. On the 10th day, the rosette shape ("budding" and expansion) of the neural tube can be observed. On the 16th day, rosette-shaped clones are gently transferred with a pipette to form neurospheres. The resulting neurospheres are suspended and cultured in neural induction medium, and the neural induction medium is replaced every other day. For compound treatment experiments, brain organoids were treated with 10 μM compounds for 7 days starting from day 30.

[0276] Dissociation of organoids for 2D culture

[0277] On day 28, neurospheres were dissociated into single cells using TrypLE (Gibco, 12604-021) and then seeded at a density of 30,000-50,000 cells / ml onto coverslips coated with poly-ornithine (Sigma, St. Louis, USA, P3655) and Matrigel (Corning, New York, USA, 354230) for further differentiation. For compound treatment experiments, cortical neurons were treated with 10 μM compounds for 7 days starting from day 30.

[0278] Preparation of recombinant human LC3B

[0279] Recombinant human LC3B was prepared and verified according to the method of WO2020182143A1.

[0280] General Assay Procedures for Treatment of Cells with Compounds

[0281] The compound was prepared as a DMSO stock solution. Before being used to treat cells, the compound stock solution was diluted with DMSO to a working solution of a specified concentration. The test compound was added to the cells respectively. The control wells were replaced with an equal volume of DMSO (blank control). The cells were treated with the compound under the conditions of a cell culture incubator cultured at 37°C with a mixed humidified atmosphere of 5% CO2 and 95% air.

[0282] Immunofluorescence staining and confocal imaging

[0283] After the compound treatment for a specified time, the cells in the multiwell plate were washed twice with phosphate buffered saline (PBS) and then fixed with 4% paraformaldehyde (PFA, Sigma, St. Louis, USA, P6148) at room temperature for 10 minutes. After washing with PBS, the cells were permeabilized in 0.5% PBST (PBS + 0.5% Triton X-100) for 15 minutes and blocked with 3% bovine serum albumin (BSA, sigma B2064) in 0.1% PBST (PBS + 0.1% Triton X-100) at room temperature for 1 hour. The cells were then incubated with the primary antibody at 4 ° C overnight and with the secondary antibody at room temperature for 1 hour. After washing three times with 0.1% PBST buffer, the cells were incubated with DAPI (Beyotime, Shanghai, China, C1002) for 10 minutes and then sealed onto a slide with Mounting Medium (Beyotime, P0128S).

[0284] For organoids, fix with 4% PFA for 2-4 hours and dehydrate with 20% and 30% sucrose solution (Sigma, V900116-500G). After dehydration, the organoids were embedded in OCT and cut into a thickness of 10 μm using a constant temperature cryostat. The slices were washed three times with PBS and blocked with PBS containing 1% Triton (Beyotime, ST795) and 5% donkey serum (Millipore, Darmstadt, Germany, S30-KC) for 1 hour at room temperature. Then, the organoids were incubated overnight with a primary antibody diluted with PBS containing 0.2% Triton and 5% donkey serum at 4 ° C. On the second day, the slices were washed three times with PBS and then incubated with a secondary antibody diluted with PBS containing 5% donkey serum for 1 hour at room temperature in the dark. Finally, the sections were washed three times with PBS and mounted on slides using Fluoromount-G mounting solution (Southern Biotech, Birmingham, USA, 0100-01).

[0285] For cortical neurons cultured on coverslips, fix with 4% PFA for 30 minutes and then wash three times with PBS. Then, the coverslips were incubated with PBS containing 0.2% Triton for 10 minutes to achieve permeabilization and blocked with 10% donkey serum for 1 hour. All coverslips were incubated overnight at 4°C with primary antibodies diluted in PBS containing 0.2% Triton and 5% donkey serum. The next day, the coverslips were washed three times with PBS, and then the coverslips were incubated with secondary antibodies diluted in PBS containing 5% donkey serum for 1 hour at room temperature in the dark. After washing the coverslips three times with PBS, the coverslips were fixed to slides.

[0286] Imaging was performed using a Nikon 80i, LSM-800 (Zeiss, Oberkochen, Germany) or Leica TCS SP8 confocal microscope system (Leica, Weztlar, Germany) with a 20X, 63X or 100X objective.

[0287] Antibodies used: FKBP (Abcam, Cambridge, UK, ab2918), TOM20 (Proteintech, Chicago, USA, 11802-1-AP) or (Abcam, ab78547), SOX2 (R&D, Minneapolis, USA, AF2018), TUJ1 (Sigma, T8660), CTIP2 (Abcam, ab18465). Secondary antibodies for immunofluorescence: Goat anti-rabbit IgG (Invitrogen, Carlsbad, USA, A11034), Goat anti-rabbit IgG (Invitrogen, A11011), Goat anti-rabbit IgG (Invitrogen, A21070), Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific, Waltham, USA, A21206), Alexa Fluor 546 goat anti-rat (Thermo Fisher Scientific, A11081), Alexa Fluor 546 donkey anti-goat IgG (Thermo Fisher Scientific, A11056), Alexa Fluor 546 donkey anti-mouse IgG (Thermo Fisher Scientific, A10036), Alexa Fluor 647 donkey anti-mouse IgG (Thermo Fisher Scientific, A31571).

[0288] Acquisition of mito-DsRed real-time images: After plasmid transfection and compound treatment for the indicated time, cells were stained with 5 μg / mL hochest 33342 (MCE New Jersey, USA, HY15559) for 30 minutes at 37° C. After washing twice with PBS, images were taken using a Leica TCS SP8 confocal microscope system with a 100X objective.

[0289] Reagents for mito-tracker staining: MitoTracker TM Red CMXRos(Invitrogen,M7512)、MitoTracker TM Deep Red FM(Invitrogen,M22426).

[0290] Sample preparation for transmission electron microscopy (TEM)

[0291] On day 37 of differentiation, the organoids were fixed with a PBS solution containing 2.5% glutaraldehyde and incubated overnight at 4°C. The glutaraldehyde solution was discarded and the organoids were rinsed four times with PBS (15 minutes each time). 1% osmium tetroxide was added and incubated with the organoids at 4°C for 2 hours. A 2% aqueous solution of uranyl acetate was added and incubated with the organoids for 2 hours. Then, dehydrated with a 50%-100% gradient of acetone. The organoids were then incubated with a mixture of 100% acetone and EPON812 resin in a 1:1 ratio for 1.5 hours. The organoids were then embedded in the embedding agent and left overnight. Ultrathin sections (0.05 μm) were prepared. The sections were stained with uranium for half an hour and with lead for 10 minutes. Finally, electron microscope images were acquired.

[0292] Colocalization

[0293] For images captured by confocal microscopy, the signal intensities of channel 1 and channel 2 within a specified distance were analyzed. The Pearson correlation coefficient (PCC) was calculated using the Coloc2 plugin of FIJI to measure the colocalization of the signals of channel 1 and channel 2. The PCC ranges from -1 to +1, where -1 indicates that the signals of channel 1 and channel 2 are completely separated, and +1 indicates that the signals of the two channels are completely colocalized.

[0294] Western blotting

[0295] Total protein of cells was extracted with 2xSDS buffer (50mM Tris pH 6.8, 2% SDS, 100mM DTT, 12.5% ​​glycerol, 0.04% bromophenol blue) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland, 04693132001), and then boiled at 95°C for 10 minutes. Proteins were separated with 4%-12% Bis-Tris Sure PAGE (Genscript, Nanjing, China, M00654) and immunoblotted with primary and secondary antibodies. Signals were detected with High-sig ECL protein blotting substrate (Thermo Fisher Scientific, 32209) and imaged with e-Blot (e-BLOT, Shanghai, China). Antibodies used: TOM20 (Proteintech, 11802-1-AP), LC3B (Abcam, ab192890), P62 (Proteintech, 18420-1-AP), UQCRC1 (Proteintech, 21705-1-A), β-tubulin (Abcam, ab6046), GFP (Ab mart, Shanghai, China, M20004L), GST (Proteintech, 66001-2-Ig), TSPO (ABclonal, Wuhan, China, A4881), Beta-Actin (Proteintech, 66009-1-Ig), Flag (Abmart, M20008).

[0296] Co-immunoprecipitation (Co-IP)

[0297] The cells were lysed in NP-40 buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) supplemented with a protease inhibitor cocktail at 4° C. for 30 minutes. The homogenate obtained was centrifuged at 13,000×g at 4° C. for 15 minutes, and the supernatant was collected.

[0298] For the detection of the interaction between FKBP-LC3B and TSPO-Flag-FRB, the supernatant was incubated with Flag magnetic beads (Bimake, Houston, USA, B26101) at 4°C on a rotary shaker overnight; for the detection of the interaction between LC3B and TSPO, the supernatant was incubated with Agarose (Chromotek, Munich, Germany, gta) was incubated overnight.

[0299] The cells were then washed four times with NP-40 buffer and eluted with 2x SDS buffer for Western blot analysis.

[0300] In vitro pull-down assay

[0301] In vitro pull-down assays were used to determine the interaction of TSPO or PARKIN with LC3B, as well as the effect of the test compound on this interaction.

[0302] The cells were lysed, wherein the lysis buffer was NP-40 buffer supplemented with a protease inhibitor cocktail, and lysed at 4°C for 30 minutes. The obtained homogenate was centrifuged at 13,000×g for 15 minutes at 4°C, and then 800 μL of the supernatant was collected and incubated with the test substance (Compound 1, Compound 2, Compound T1) or DMSO and 10 μg of purified GST-LC3B protein at 4°C for 10 hours, and then 10 μL of GST-tagged protein purification magnetic beads (CST, 11847S) were added to the mixture and incubated overnight at 4°C.

[0303] For the detection of the interaction between purified 6*His-TSPO and GST-LC3B, 1 μg 6*His-TSPO and 2 μg GST-LC3B were incubated with the compound at 4°C for 8 hours. TSPO antibody was added and incubated overnight at 4°C. Protein G magnetic beads (ThermoFisher Scientific, 88847) were added to the mixture and incubated at 4°C for 6 hours.

[0304] After incubation, the beads were washed four times with NP-40 buffer and then eluted in 2x SDS buffer for Western blot analysis.

[0305] mito-keima detection

[0306] Keima (UniProt ID: Q1JV70) is a dual-excitation fluorescent protein that is pH-sensitive and relatively resistant to lysosomal proteases. Keima has a shorter excitation wavelength (440 nm, green) at neutral pH and a longer excitation wavelength (586 nm, red) at acidic pH. Mito-keima was targeted to the mitochondrial matrix using a mitochondrial targeting sequence from COX VIII. Using the properties of mito-keima, it is possible to understand whether the mitochondria it marks are in physiological conditions (PH 8.0) or have entered the lysosome (PH 4.5), and the level of mitophagy can be detected.

[0307] After the cells were transfected with mito-keima for a specified time, they were treated with the compound. Phase contrast and red fluorescence images in the cell culture chamber were captured using IncuCyte technology (Essen Bioscience, Ann Arbor, USA, IncuCyte ZOOM). Photos were taken every 3 hours, and four different fields of view were taken for each well. Quantification was performed using IncuCyte 2011A software.

[0308] Mitochondrial DNA copy number detection

[0309] For cells in multi-well plates, genomic DNA was extracted using the Ezup column animal genomic DNA extraction kit according to the manufacturer's instructions. The obtained DNA was subjected to quantitative real-time PCR using the QuantStudioTM 7Flex Real-Time PCR System (LifeTechnologies) to determine the expression of the gene of interest. The mRNA level of the target gene was normalized using β2 microglobulin as an internal reference. The qRT-PCR primers are shown in the following table:

[0310]

[0311] Preparation Example 1 N-(10-(3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-2-oxoindolyl-1-yl)decane 2-(2-phenyl-1H-indol-3-yl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide (Compound 1)

[0312]

[0313] Step 1: 2-Oxo-2-(2-phenyl-1H-indol-3-yl)acetyl chloride

[0314] A solution of 2-phenyl-1H-indole (500 mg, 2.59 mmol) in anhydrous tetrahydrofuran (10 mL) was bubbled with nitrogen for 5 minutes, and then oxalyl chloride (442 mg, 3.11 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 1 hour. After TLC showed that the reaction was complete, the reaction mixture was concentrated to obtain 2-oxo-2-(2-phenyl-1H-indol-3-yl)acetyl chloride as a red oil (600 mg, yield 82%), which was used directly in the next step.

[0315] Step 2: N-(10-hydroxydecyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide

[0316] 2-Oxo-2-(2-phenyl-1H-indol-3-yl)acetyl chloride (520 mg, 1.84 mmol) and triethylamine (743 mg, 7.36 mmol) were dissolved in dichloromethane (15 mL) to obtain a solution. 10-Aminodecane-1-ol (318 mg, 1.84 mmol) was dissolved in dichloromethane (10 mL), cooled to 0°C, and then added dropwise to the above solution. The mixture was stirred at room temperature for 1 hour and concentrated. The residue was purified by flash column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give N-(10-hydroxydecyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide as a white solid (540 mg, yield 70.1%).

[0317] Step 3: N-(10-bromodecyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide

[0318] N-(10-hydroxydecyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide (520 mg, 1.24 mmol), N-bromosuccinimide (242 mg, 1.36 mmol) and triphenylphosphine (487 mg, 1.86 mmol) were added to dichloromethane (20 mL). The mixture was stirred at room temperature for 2 hours. After the reaction was completed, water (40 mL) was added to the reaction mixture and extracted with dichloromethane (40 mL×2). The organic layers were combined and concentrated, and the residue was purified by flash column chromatography (dichloromethane / methanol=5 / 1) to obtain N-(10-bromodecyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide as a white solid (500 mg, yield 83.9%).

[0319] Step 4: Compound 1

[0320] 3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodoindoline-2-one (99 mg, 0.19 mmol), N-(10-bromodecyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide (100 mg, 0.19 mmol), potassium iodide (31.5 mg, 0.19 mmol) and cesium carbonate (123.9 mg, 0.38 mmol) were added to N,N-dimethylformamide (10 mL). The mixture was stirred at 50 ° C for 3 hours. The reaction mixture was cooled to room temperature, diluted with water (60 mL), and extracted with ethyl acetate (30 mL×2). The organic layers were combined, then dried and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1-1 / 1) to give N-(10-(3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-2-oxoindolin-1-yl)decyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide (Compound 1) as a yellow solid (23 mg, yield 13%).

[0321] Among them, compound L1 (3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodoindoline-2-one) is commercially available and can also be prepared according to the following method:

[0322]

[0323] Step 1: 5-iodoindolin-2-one

[0324] Indolin-2-one (3.0 g, 22.5 mmol) and NIS (6.1 g, 27.0 mmol) were dissolved in acetic acid (30 mL). The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated by rotary evaporator to remove most of the solvent, and the obtained residue was slurried with ethyl acetate, filtered, and the filter cake was rinsed with a small amount of ethyl acetate and dried to obtain the product red solid 5-iodoindolin-2-one (4.6 g, yield: 79.3%). MS m / z (ESI): 259.9 [M+H] +

[0325] Step 2: Compound L1 (3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodoindolin-2-one)

[0326] Dissolve 5-iodoindoline-2-one (2.0 g, 7.72 mmol) in ethanol (20 mL), add 3,5-dibromo-4-hydroxybenzaldehyde (2.16 g, 7.72 mmol) and piperidine (131 mg, 1.54 mmol). The mixture was reacted at 80 ° C for 16 hours. The reaction solution was cooled to room temperature, filtered, and the filter cake was rinsed with ether and petroleum ether respectively, and dried to obtain a yellow solid compound L1 (1.8 g, 45.0%). MS m / z (ESI): 521.7 [M+H] +

[0327] Preparation Example 2 N-(10-((7-hydroxy-2-oxo-4-phenyl-2H-chromen-5-yl)oxy)decyl)-2-oxo- 2-(2-Phenyl-1H-indol-3-yl)acetamide (Compound 2)

[0328]

[0329] 5,7-dihydroxy-4-phenyl-2H-coumarin (80 mg, 0.315 mmol) and N-(10-bromodecanyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide (151.2 mg, 0.315 mmol) obtained in step 3 of Preparation Example 1 were dissolved in N,N-dimethylformamide (4 mL) to obtain a solution, and potassium carbonate (260.8 mg, 1.89 mmol) was then added. The mixture was stirred at 60 ° C for 16 hours. The reaction mixture was cooled to room temperature, diluted with water (30 mL), and extracted with ethyl acetate (30 mL×2). The organic layers were combined, dried over sodium sulfate and concentrated. The residue was purified by preparative HPLC to give N-(10-((7-hydroxy-2-oxo-4-phenyl-2H-chromen-5-yl)oxy)decyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide (Compound 2) as a red solid (22.3 mg, yield 11%).

[0330] Example 1 Compound 1 promotes mitochondria to enter lysosomes in cells

[0331] 1.1 mito-keima detection

[0332] Preliminary test: Flag-mito-keima detection

[0333] To verify that compound 1 does not affect the expression of mito-keima, HeLa cells were transfected with a mito-keima plasmid with a Flag tag (Flag-mito-keima), and then the following tests were performed:

[0334] (1) Cells were treated with compound 1 (20 μM), CQ (20 μM), or compound 1 (20 μM) + CQ (20 μM) for 48 hours. Western blot detection ( Figure 1 ).

[0335] (2) Cells were treated with compound 1 (20 μM) for 48 h. Images were captured using a confocal microscope, and the red signal of Flag-mito-keima was quantified ( Figure 2 ).

[0336] (3) Cells were treated with compound 1 (20 μM), CQ (20 μM), or compound 1 (20 μM) + CQ (20 μM) for 48 h. Images were captured using Incucyte, and the counts and intensities of the Flag-mito-keima red signal were quantified ( Figure 3 ).

[0337] The results showed that (1) above proved that compound 1 did not affect the expression of Flag-mito-keima; and (2) and (3) above proved that compound 1 enhanced the red signal of Flag-mito-keima in HeLa cells, confirming that the Flag tag did not affect the function of mito-keima and the purpose of the experiment.

[0338] mito-keima detection:

[0339] According to the method described above, HeLa cells were transfected with mito-keima, and then treated with compounds. The pH sensitivity of mito-keima was used to detect the entry of mitochondria into acidic lysozyme during mitophagy by analyzing the percentage of mito-keima red signal positive area. The compound was one of the following: compound 1, compound 2, carbonyl cyanide m-chlorophenylhydrazone (CCCP), LC3 ligand compound L1, and TSPO ligand compound T1.

[0340] The structures of compound L1 (Li et al., "Allele-selective lowering of mutant HTT protein by HTT–LC3 linker compounds." Nature, 2019, 575, pp 203–209) and compound T1 (Rupprecht et al., "Translocator protein (18 kDa) (TSPO) as a therapeutic target for neurological and psychiatric disorders." Nature Reviews Drug Discovery, 2010, 9, pp 971-988) are as follows:

[0341]

[0342] According to the above method:

[0343] The cells were treated with compound 1 (20 μM), compound 2 (20 μM), CCCP (5 μM) and compound T1 (20 μM) and the above assay was performed. The results are shown in Figure 4 The above test was performed with compound 1 (20 μM), CCCP (10 μM) and compound L1 (20 μM). The results are shown in Figure 5 shown. Figure 4 and Figure 5 The results showed that the use of TSPO ligand compound T1 or LC3 ligand compound L1 alone had no effect on the mito-keima red signal. However, among the two chimeric compounds containing mitochondrial binding parts and LC3 binding parts, compound 1 increased the mito-keima red signal, while compound 2 did not.

[0344] The cells were treated with 10, 15 or 20 μM of compound 1 and the above test was performed. It was found that compound 1 increased the mito-keima red signal in a dose-dependent manner within the μM range ( Figure 6 ).

[0345] The cells were treated with compound 1 (20 μM), CCCP (10 μM), compound L1 (20 μM) and compound T1 (20 μM) for 72 hours and the above test was performed. Figure 7 The cells were treated with compound 1 (20 μM) and CCCP (5 μM) for 48 hours and the above test was performed. Figure 8 shown. Figure 7 and Figure 8 The results showed that compound 1 not only increased the red signal of mito-keima, but also changed the shape of the particles detected by mito-keima into a round shape, similar to the situation after CCCP treatment. This may be due to the entry of mitochondria into lysosomes.

[0346] 1.2 Colocalization detection of TOM20 and LAMP1

[0347] 293T cells were transfected with LAMP1-mCherry and then treated with compound 1 (20 μM) for 48 hours. Immunofluorescence detection was performed with TOM20 antibody, and images were captured with a confocal microscope. The signal intensities of TOM20 and LAMP1 within a specified distance were analyzed, and the Pearson correlation coefficient was calculated. The results showed that compound 1 enhanced the colocalization of the mitochondrial marker mitochondrial outer membrane protein TOM20 and the lysosomal marker LAMP1, and that this promotion could be inhibited in the presence of the autophagy inhibitor CQ (i.e., chloroquine); further confirming that compound 1 promoted the entry of mitochondria into lysosomes ( Fig. 9 ).

[0348] The results demonstrated that compound 1 could promote the entry of mitochondria into lysosomes.

[0349] Example 2 Compound 1 promotes mitochondrial degradation in HeLa cells

[0350] 2.1 mito-DsRed detection

[0351] According to the method described above, HeLa cells were transfected with mito-DsRed to obtain cells overexpressing mito-DsRed, and then the cells were treated with compound 1 (20 μM), compound T1 (20 μM), or compound L1 (20 μM) for 48 hours. Images were collected using a confocal microscope. The results showed that compound 1 significantly reduced the mito-DsRed positive area and signal intensity ( Fig.10 ), indicating that compound 1 promoted mitochondrial degradation.

[0352] 2.2 Detection of mitochondrial protein levels

[0353] HeLa cells were treated with 2, 5, 10 or 20 μM of compound 1 for 48 h. The TOM20 level was detected by immunofluorescence as described above. Fig.11 As shown. HeLa cells were treated with 2, 5, 10 or 20 μM compound 1 for 72 hours and then the cells were collected. The ratio of TOM20 or UQCRC1 / actin was quantified by Western blot analysis as described above. The results are shown in Fig.12 As shown. HeLa cells were treated with 20 μM compound 1 for 24 hours or 48 hours, or with 10 μM CCCP for 48 hours. The TOM20 level was detected by immunofluorescence according to the method described above. The results are shown in Fig.13 The results demonstrated that compound 1 reduced the levels of endogenous mitochondrial outer membrane protein TOM20 and inner membrane protein UQCRC1 in a dose- and time-dependent manner.

[0354] 2.3 Detection of mitochondrial DNA levels

[0355] HeLa cells were treated with 20 μM compound 1 for 72 h. tRNA-Leu (UUR) and ND5 DNA levels were analyzed by qPCR to determine mtDNA levels ( Fig.14 ). The results demonstrated that compound 1 reduced mtDNA levels.

[0356] The results showed that compound 1 could induce mitochondrial degradation.

[0357] 2.4 CCCP enhances the promoting effect of compound 1 on mitochondrial degradation

[0358] HeLa cells were transfected with mito-keima as described above, and then treated with compound 1 (20 μM), CCCP (1 μM), or compound 1 (20 μM) + CCCP (1 μM).

[0359] 1μM CCCP depolarized the mitochondrial membrane but did not induce mitophagy. The experimental results showed that the promoting effect of compound 1 on mitophagy was enhanced by 1μM CCCP ( Fig.15 ). This indicates that compound 1 preferentially degrades damaged mitochondria.

[0360] Example 3 Compound 1 promotes mitochondrial degradation in HEK293-αsyn-A53T cells

[0361] The effect of compound 1 on mitochondrial degradation in a PD-related cell model (HEK293 cells stably expressing α-synuclein A53T (HEK293-αsyn-A53T)) was investigated.

[0362] HEK293-αsyn-A53T cells were treated with 2, 5, 10 or 20 μM of compound 1 for 48 hours. The TOM20 level was detected by immunofluorescence as described above ( Fig.16 ).

[0363] HEK293-αsyn-A53T cells were treated with 2, 5, 10, or 20 μM compound 1 for 24 or 48 h, and then cells were collected for Western blot analysis and the ratio of TOM20 or UQCRC1 / actin was quantified ( Fig.17 ).

[0364] The results showed that compound 1 reduced the levels of TOM20 and UQCRC1 in HEK293-αsyn-A53T cells.

[0365] Example 4 Compound 1 specifically promotes mitochondrial degradation

[0366] In order to exclude the possibility that compound 1 promotes mitochondrial autophagy by destroying mitochondria, this example studies the effect of compound 1 on mitochondrial membrane potential (MMP) and cells.

[0367] 4.1 Compound 1 does not affect mitochondrial membrane potential

[0368] HeLa cells were treated with compounds for 2 hours and then stained with TMRE dye (2 μM). Images were captured by confocal microscopy and TMRE intensity was quantified to measure mitochondrial membrane potential (MMP) in HeLa cells. The compound was one of the following: compound 1 (20 μM), compound T1 (20 μM), compound L1 (20 μM), positive control CCCP (10 μM). The results are shown in Fig.18As shown, Compound 1, Compound T1, and Compound L1 did not affect MMP at least at the concentrations tested. In contrast, CCCP depolarized the mitochondrial membrane as expected.

[0369] 4.2 Compound 1 does not cause cytotoxicity

[0370] HeLa cells were treated with 5, 10 or 20 μM compound 1 for 24 or 48 hours, and then the cytotoxicity of compound 1 was determined using Cell Counting Kit-8 (CCK8) (Dojindo, Kumamoto Prefecture, Japan, CK04) according to the manufacturer's instructions. The results showed that compound 1 did not cause cytotoxicity ( Fig.19 ).

[0371] 4.3 Compound 1 does not affect the overall autophagy level

[0372] HeLa cells were treated with 20 μM compound 1 for 24 h, and then cells were collected for Western blot analysis and quantification of the ratio of LC3BII / LC3BI or p62 / tubulin. The results showed that compound 1 treatment did not change SQSTM1 / p62 or LC3BII / LC3BI ( Fig. 20 ), indicating that the overall autophagy level was not affected.

[0373] The results demonstrated that compound 1 specifically promoted mitochondrial degradation rather than reducing mitochondrial levels by damaging mitochondria or cells.

[0374] Example 5 Compound 1 induces mitochondrial degradation via LC3- and ATG-dependent autophagy

[0375] 5.1 Compound 1 enhances the colocalization of LC3 and mitochondria

[0376] HeLa cell lines stably expressing PMSCV-GFP-LC3B were transfected with mito-DsRed, and then treated with compound 1 (20 μM), CQ (20 μM), or compound 1 (20 μM) + CQ (20 μM) for 48 hours. Images were captured using a confocal microscope. The signal intensities of Mito-DsRed and GFP-LC3B within the specified distance were analyzed, and the Pearson correlation coefficient was calculated. The results showed that compound 1 enhanced the colocalization of LC3 and mitochondria, and this promotion was further enhanced in the presence of the autophagy inhibitor CQ (i.e., chloroquine) ( Fig.21 The further enhancement of colocalization may be due to the fact that CQ inhibits the degradation of colocalized LC3-mitochondria.

[0377] 5.2 Effect of LC3B knockout on the action of compound 1

[0378] WT and LC3B KO 293T cells were stained with mito-tracker-red and then treated with compound 1 (10 μM) for 48 h. Images were captured using confocal microscopy, and the percentage of mito-tracker-positive area was quantified. Fig. 22 Compound 1 was able to dose-dependently reduce mito-tracker in WT 293T cells, but for LC3B KO 293T cells, compound 1 was no longer able to reduce the percentage of mito-tracker-positive area.

[0379] WT or LC3B KO 293T cells were treated with 20 μM compound 1 for 48 hours, and then the effect of compound 1 on TOM20 levels was detected by immunofluorescence using a method similar to Example 2.2 ( Fig.23 ).

[0380] WT or LC3B KO 293T cells were treated with 5 μM, 10 μM or 20 μM compound 1 for 72 hours, and then the effect of compound 1 on TOM20 level was detected by Western blotting using a method similar to Example 2.2 ( Fig.24 ).

[0381] Fig.23 and Fig.24 Compound 1 was able to reduce TOM20 levels in WT 293T cells, but for LC3BKO 293T cells, compound 1 was no longer able to reduce TOM20.

[0382] Therefore, it can be seen that compound 1 must act through LC3.

[0383] 5.3 Effect of ATG5 knockdown on the effect of compound 1

[0384] Autophagy-related protein 5 (ATG5) is associated with LC3 lipidation and plays a key role in autophagy.

[0385] HeLa cell lines stably expressing PMSCV-GFP-LC3B were transfected with ATG5 siRNA. The cells were then treated with compound 1 (20 μM), CQ (20 μM), or compound 1 (20 μM) + CQ (20 μM) for 48 hours. The effect of compound 1 on the colocalization of GFP-LC3B and TOM20 was then detected using a method similar to Example 1.1 ( Fig.25 ).

[0386] HeLa cells were transfected with mito-keima and ATG5 siRNA and then treated with compound 1 (20 μM). The effect of compound 1 on the red signal of mito-keima was then detected using a method similar to that in Example 1.1 ( Fig.26 ).

[0387] HeLa cells were transfected with ATG5 siRNA and then treated with compound 1 (20 μM) for 48 hours. The effect of compound 1 on TOM20 levels was then detected by immunofluorescence using a method similar to that in Example 2.2 ( Fig. 27 ).

[0388] WT or ATG5 KO 293T cells were treated with compound 1 (20 μM) for 48 hours. Then, the effect of compound 1 on TOM20 level was detected by immunofluorescence using a method similar to that in Example 2.2 ( Fig.28 ).

[0389] WT or ATG5 KO 293T cells were treated with compound 1 (20 μM) for 72 hours. The effect of compound 1 on the levels of TOM20 and UQCRC1 was then detected by Western blotting using a method similar to that in Example 2.2 ( Fig.29 ).

[0390] The results showed that for cells with ATG5 knockdown or knockout: compound 1 could no longer enhance the colocalization of LC3B and TOM20; it could no longer increase the mito-keima red signal; and the effect of reducing TOM20 and UQCRC1 was partially abolished.

[0391] 5.4 Effect of ATG9A knockout on the action of compound 1

[0392] According to the literature (Pan et al., "Gelation of cytoplasmic expanded cag rna repeats701suppresses global protein synthesis." Nature chemical biology, 2023) method, STHdh knockout ATG9A was prepared. Q7 / Q7 WT or ATG9A KO STHdh cells were treated with compound 1 (20 μM). Q7 / Q7 The cells were then incubated for 72 hours. The effect of compound 1 on the TOM20 level was then detected by Western blotting using a method similar to that in Example 2.2 ( Fig.30 The results showed that for cells with ATG9A knockout, the effect of compound 1 on reducing TOM20 and UQCRC1 was abolished, and was more complete than that of ATG5 knockout.

[0393] In conclusion, compound 1 induced mitochondrial degradation that depended on LC3 and ATG proteins, and the mechanism was autophagy.

[0394] Example 6 Compound 1 induces mitochondrial degradation via the autophagy-lysosome pathway

[0395] 6.1 Autophagy inhibitors eliminate compound 1-induced mitochondrial degradation

[0396] In this example, the effect of compound 1 on mitochondria was observed in the presence of the autophagy inhibitor CQ.

[0397] HeLa cells or 293T cells were transfected with mito-keima, and then the cells were treated with the compound. Then, the effect of the compound on the red signal of mito-keima in HeLa cells and 293T cells was detected by a method similar to Example 1.1; the results are shown in FIG. Fig.31a and 31b As shown;

[0398] HeLa cells were transfected with mito-DsRed and then treated with the compound for 48 hours. The effect of the compound on the mito-DsRed signal intensity was then detected using a method similar to Example 2.1; the results are shown in Fig.32 As shown;

[0399] The 293T cells were treated with the compound for 48 hours, and then the effect of the compound on the TOM20 level was detected by immunofluorescence using a method similar to Example 2.2; the results are shown in Fig.33 As shown;

[0400] HeLa cells were treated with the compound for 72 hours. The effect of the compound on TOM20 levels was then detected by Western blotting using a method similar to Example 2.2; the results are shown in Fig.34 As shown;

[0401] The effect of the compound on mtDNA level in HeLa cells was detected by a method similar to that in Example 2.3; the results are shown in Fig.35 As shown;

[0402] Wherein, the compound is compound 1 (20 μM), CQ (20 μM) or compound 1 (20 μM) + CQ (20 μM).

[0403] The results showed that in the presence of CQ: compound 1 could no longer increase the mito-keima red signal; could no longer decrease mito-DsRed; could no longer decrease TOM20; and could no longer decrease mtDNA.

[0404] 6.2 Mito-QC Testing

[0405] GFP fluorescence is quenched in acidic lysosomes. Based on this, mito-QC (mito-mCherry-GFP) detection was used to visualize mitochondria.

[0406] HeLa cells were transfected with mito-mCherry-GFP and then treated with compound 1 (20 μM), CQ (20 μM), or compound 1 (20 μM) + CQ (20 μM) for 48 h, and images were captured using a confocal microscope. The red signal intensity or the ratio of (red signal - green signal) / red signal of each group was quantified. The results are shown in Fig.36 As shown. By calculating the (red signal - green signal) / red signal of mito-QC, it is shown that mitochondrial autophagy is enhanced, indicating that compound 1 will increase the ratio of lysosomes to mitochondria. CQ can eliminate this effect.

[0407] The results demonstrated that compound 1-promoted mitochondrial degradation depended on the autophagy-lysosome pathway.

[0408] Example 7 Compound 1-induced mitochondrial degradation is dependent on TSPO

[0409] This example verifies that compound 1 exerts its effect through TSPO.

[0410] HeLa cell lines stably expressing PMSCV-GFP-LC3B were transfected with mito-DsRed and TSPO siRNA, and then treated with compound 1 (20 μM), CQ (20 μM), or compound 1 (20 μM) + CQ (20 μM) for 48 hours. The effect of compound 1 on the colocalization of mito-DsRed and GFP-LC3B was then detected using a method similar to Example 5.1. The results are shown in Figure 5. Fig.37 As shown. HeLa cells were transfected with mito-keima and TSPO siRNA, and then treated with compound 1 (20 μM). The effect of compound 1 on the red signal of mito-keima was then detected using a method similar to Example 1.1. The results are shown in Fig.38 As shown. HeLa cells were transfected with TSPO siRNA and then treated with compound 1 (20 μM) for 48 hours. The effect of compound 1 on TOM20 levels was then detected by immunofluorescence using a method similar to Example 2.2. The results are shown in Fig.39 As shown. HeLa cells were transfected with TSPO siRNA and then treated with compound 1 (20 μM) for 72 hours. The effect of compound 1 on TOM20 levels was then detected by Western blotting using a method similar to Example 2.2. The results are shown in Fig.40 The results showed that for cells with TSPO knockdown: the promoting effect of compound 1 on the co-localization of mitochondria (mito-DsRed) and LC3B was significantly reduced, and it could no longer increase the mito-keima red signal; it could no longer reduce the mitochondrial protein.

[0411] The results confirmed that compound 1 targets mitochondria through TSPO to promote mitochondrial degradation.

[0412] Example 8 Compound 1 enhances the interaction between TSPO and LC3B

[0413] 8.1 Pull down

[0414] Pull-down tests were performed as described above.

[0415] HeLa cell lysates were incubated with 20 μM or 100 μM compound 1 and GST-LC3B, and then pulled down with GST-tagged protein purification magnetic beads for western blot analysis. Fig.41a As shown. HeLa cell lysates were incubated with 100 μM compound 1, 100 μM compound L1, or 100 μM compound T1 and GST-LC3B protein, and then pulled down with GST-tagged protein purification magnetic beads for western blot analysis. The results are shown Fig.41b shown.

[0416] The results showed that compound 1 promoted the interaction between GST-LC3B and TSPO in a dose-dependent manner, while compound L1 and compound T1 had no such promoting effect.

[0417] 8.2 Co-immunoprecipitation

[0418] According to the method described above, purified TSPO with 6*His was incubated with 20μM or 100μM compound 1 and purified GST-LC3B, and then immunoprecipitated with TSPO antibody. The results showed that compound 1 promoted the interaction between 6*His-TSPO and GST-LC3B ( Fig.42 ). HeLa cell lines stably expressing PMSCV-GFP-LC3B were treated with compound 1 (20 μM), CQ (20 μM), or compound 1 (20 μM) + CQ (20 μM), and then immunoprecipitated with anti-GFP magnetic beads ( Fig.43 ). The results showed that compound 1 promoted the interaction between LC3B and TSPO. Interestingly, as mentioned above Fig.41a and 41b As shown, compound 1 did not affect the interaction between SQSTM1 / p62 and LC3B, indicating that compound 1 may not affect receptor-based selective autophagy and specifically enhance LC3B-TSPO interaction.

[0419] Example 9 Compound 1 enhances intracellular colocalization of endogenous mitochondria and LC3B

[0420] As mentioned above Fig.21 As shown, by detecting the colocalization of mito-DsRed and LC3B, it has been demonstrated that compound 1 can bind to mitochondria and LC3.

[0421] HeLa cell lines stably expressing PMSCV-GFP-LC3B were treated with compound 1 (20 μM), CQ (20 μM), or compound 1 (20 μM) + CQ (20 μM). The colocalization of GFP-LC3B and TSPO was detected by immunofluorescence using TSPO antibody according to the method described above ( Fig.44 ). The results showed that compound 1 enhanced the colocalization between LC3B and TSPO, and this promoting effect was further enhanced in the presence of CQ.

[0422] Example 10 Compound 1 attenuates MPP in a cell PD model + Induced toxicity

[0423] 1-Methyl-4-phenylpyridinium (MPP + ) to treat SH-SY5Y cells, and a PD-related cell model can be prepared. + This leads to PD-like pathology, including depolarization of mitochondrial membrane potential, increased reactive oxygen species, and caspase activation.

[0424] 10.1 Compound 1 inhibits the MPP in a cell PD model + Induced apoptosis

[0425] SH-SY5Y-αsyn-WT or SH-SY5Y-αsyn-A53T cells were treated with compound 1 (10 μM or 20 μM) for 72 hours, and then the effect of compound 1 on TOM20 levels was detected by Western blotting using a method similar to Example 2.2. The results are shown in Fig.45 and Fig.46 shown.

[0426] Using MPP + (500 μM) or compound 1 (10 μM or 20 μM) + MPP + SH-SY5Y-αsyn-WT or SH-SY5Y-αsyn-A53T cells were treated with 500 μM (500 μM) for 48 hours, and then the effect of compound 1 on TOM20 level was detected by Western blotting using a method similar to Example 2.2. The results are shown in Fig.47 and Fig.48 shown.

[0427] The results showed that compound 1 treatment promoted MPP + Mitochondrial degradation in treated SH-SY5Y-αsyn-WT and SH-SY5Y-αsyn-A53T cells.

[0428] 10.2 Caspase-3 Detection

[0429] SH-SY5Y-αsyn-WT and SH-SY5Y-αsyn-A53T cells were stained with NucView 488 caspase-3 dye (Biotium, Bay Area, USA, 30029) and then treated with compound 1 (10 μM or 20 μM), MPP + (500 μM) or compound 1 (10 μM or 20 μM) + MPP + Cells were treated with 500 μM of caspase-3. Phase contrast and green fluorescence images were captured in the cell culture incubator using Incucyte. Images were taken every 3 hours, and four different fields of view were taken for each well. Caspase-3 intensity and counts were quantified using IncuCyte 2011A software.

[0430] The results of Caspase-3 activation probe assay also showed that compound 1 inhibited the activation of MPP + Induced apoptosis ( Fig.49a , 49b and Fig.50a , 50b ).

[0431] The results demonstrate the potential rescue effect of compound 1 on neurodegenerative disease-related phenotypes.

[0432] Example 11 Compound 1 rescues relevant phenotypes in DS model

[0433] To investigate the potential rescue effect of Compound 1 on DS-related phenotypes, this example tested whether Compound 1 could improve the number of mitochondria in iPSC-derived cortical neurons and brain organoids from Down syndrome (DS) patients.

[0434] Test methods such as Fig.51 Shown: iPSCs from patients were induced to differentiate into cortical neurons and brain organoids by standard protocols. Compound 1 (10 μM) was added to the culture medium on day 30 and treated for 7 days, and then the following tests were performed.

[0435] 11.1 Effects of Compound 1 on Abnormal Distribution of Mitochondria in Cortical Neurons and Brain Organoids

[0436] Cortical neurons of the euploid (IMR90-4) and triploid (2DS3) groups were immunostained, and representative immunostaining images are shown in Figure 2. Fig.52 As shown, the analysis is Fig.53 As shown. There are more aggregated mitochondria in DS cortical neurons than in control neurons. The results showed that compound 1 alleviated the mitochondrial aggregation distribution in cortical neurons of the triploid (DS) group.

[0437] Representative immunostaining images of brain organoids are shown in Figure 2. Fig.54The percentage of cells with aggregated mitochondria in the Sox2-positive ventricular zone of euploid and triploid brain organoids was quantified. Fig.55 The results showed that compound 1 could improve the aggregated mitochondria in DS brain organoids.

[0438] 11.2 Effects of Compound 1 on Mitochondrial Morphology in Brain Organoids

[0439] Transmission electron microscopy (TEM) images of euploid and triploid brain organoids were obtained according to the method described above. Representative TEM images are shown in Fig.56 The results showed that mitochondrial fragmentation was observed in triploids, and compound 1 improved this situation.

[0440] The average mitochondrial area in euploid and triploid brain organoids was quantified. The results showed that the average mitochondrial area in the triploid group was approximately 0.1 μm 2 , while the triploid group treated with compound 1 was about 0.2 μm 2 , similar to the euploid group ( Fig.57 ).

[0441] 11.3 Compound 1 for CTIP2 + The impact of neurons

[0442] Deep cortical neurons (CTIP2) were detected in brain organoids by immunostaining according to the method described above. + Neurons), representative immunostaining images obtained are shown in Fig.58 CTIP2 expression in the neural tube-like region of euploid and triploid brain organoids + The percentage of neurons was quantified and the results showed that compound 1 increased the expression of CTIP2 in the triploid group. + Neuron ( Fig.59 ).

[0443] The results demonstrated that compound 1 was able to correct the abnormal distribution and morphology of mitochondria in DS cortical neurons and brain organoids, demonstrating the potential rescue effect of compound 1 on DS-related mitochondrial defects.

[0444] Comparative Example 1 Effect of AUTAC4 on Mitochondria

[0445] Takahashi et al. reported a chimeric compound AUTAC4 that can specifically promote mitochondrial degradation. The structure of AUTAC4 is as follows, containing a portion targeting selective autophagy, a linker portion, and compound T1 as a portion targeting mitochondria (Takahashi et al., "AUTACs: Cargo-Specific Degraders Using Selective Autophagy." Molecular Cell, 2019, 76 (5), pp 1-14).

[0446]

[0447] AUTAC4 was prepared according to the method described in the report.

[0448] HEK293-αsyn-A53T cells or SH-SY5Y cells were stained with mito-tracker-red and then treated with compound 1 (20 μM) or AUTAC4 (20 μM) for 10 h. CCCP (40 μM) was added and treated for another 2 h. Images were captured using a confocal microscope and the mito-tracker signal intensity was quantified. Fig.60 HEK293-αsyn-A53T cells were stained with mito-tracker-green or mito-tracker-red and then treated with AUTAC4 (20 μM) for 24 h. Images were captured using a confocal microscope and the mito-tracker signal intensity was quantified. Fig.61 HeLa cells were treated with AUTAC4 (20 μM) for 72 h, and then the cells were collected for Western blot analysis and the ratio of TOM20 or UQCRC1 / actin was quantified. Fig.62 As shown. HeLa cells were transfected with mito-keima, and then treated with compound 1 (20 μM) or AUTAC4 (20 μM). The effect of compound 1 on the red signal of mito-keima was then detected using a method similar to Example 1.1. The results are shown in Fig.63 shown.

[0449] The results showed that, compared with compound 1, AUTAC4 had almost no effect on mitochondria, or at least had a much weaker effect than compound 1 on mitochondria when compared under the same experimental conditions.

[0450] The experimental results of AUTAC4 under the conditions of the present invention indicate that compounds with different mechanisms of action may play different roles in different systems, and therefore, it is advantageous to develop multiple strategies to promote mitochondrial degradation.

[0451] Comparative Example 2 Targeting PARKIN to mitochondria does not induce mitophagy

[0452] This comparative example uses the FKBP-FRB system to recruit autophagy-related proteins to mitochondria for comparison with the present invention which binds compounds via autophagosomes.

[0453] 2.1 Targeting LC3B to mitochondria using the FKBP-FRB system promotes mitochondrial degradation

[0454] The mechanism by which the FKBP-FRB system promotes the interaction between LC3B and TSPO is as follows Fig.64 As shown, LC3B was fused to FKBP, TSPO was fused to FRB, and the interaction between FKBP and FRB was induced by the dimerization inducer rapamycin.

[0455] 293T cells were transfected with FKBP-LC3B and TSPO-Flag-FRB plasmids and then treated with the indicated concentrations of dimerization inducers for 6 hours. Anti-Flag magnetic beads were then used to pull down for Western blot analysis. The results showed that the dimerization inducer promoted the interaction between LC3B and TSPO. Fig.65 As shown. 293T cells were transfected with FKBP-LC3B and TSPO-Flag-FRB plasmids. Then they were treated with a dimerization inducer (1 μM) for 24 hours. The cells were stained with mito-tracker-deep-red. A method similar to Example 5.1 was then used to detect the effect of the dimerization inducer on the colocalization of LC3B and mito-tracker. Images were captured using a confocal microscope. The mito-tracker signal intensity was quantified. The signal intensity of LC3B and mito-tracker within a specified distance was analyzed. The results are shown in Fig.66 As shown in Figure 3, dimerization inducers reduced the mito-tracker signal intensity and enhanced the colocalization of FKBP-LC3B with mitochondria. In the presence of the autophagy inhibitor CQ (20 μM), the promoting effect of dimerization inducers on colocalization was further enhanced.

[0456] 293T cells were transfected with FKBP-LC3B and Flag-TSPO or TSPO-Flag-FRB plasmids. Then, dimerization inducers were treated for 48 hours at a specified concentration. Western blotting and immunofluorescence were performed using a method similar to that in Example 2.2, and it was observed that dimerization inducers could reduce TOM20 levels ( Fig.67 and Fig.68 ).

[0457] The results showed that dimerization inducers could promote mitochondrial degradation.

[0458] The results confirmed that inducing LC3B-mitochondria interaction can enhance mitophagy.

[0459] 2.2 Targeting PARKIN to mitochondria using the FKBP-FRB system fails to promote mitochondrial degradation

[0460] It is known that the recruitment of PARKIN to mitochondria leads to the ubiquitination of OMM proteins and induces mitochondrial autophagy (Narendra et al., "Parkin is recruited selectively to impaired mitochondria and promotes their autophagy." Journal of Cell Biology, 2008, 183, pp 795-803). This comparative example adopts a method similar to that of comparative example 2.1, using TSPO-Flag-FRB and FKBP-EGFP-PARKIN, and using the FKBP-FRB system to recruit PARKIN to mitochondria to test whether it can enhance mitochondrial autophagy.

[0461] 293T cells were transfected with FKBP-EGFP-PARKIN and TSPO-Flag-FRB, and then treated with the indicated concentrations of dimerization inducers for 6 hours. Anti-Flag magnetic beads were then used to pull down for Western blot analysis. The results showed that the dimerization inducer promoted the interaction between PARKIN and TSPO ( Fig.69 ).

[0462] 293T cells were transfected with FKBP-EGFP-PARKIN and Flag-TSPO or TSPO-Flag-FRB, and then treated with a dimerization inducer (1 μM) for 24 hours. The cells were stained with mito-tracker-deep-red. The effect of the dimerization inducer on the colocalization of FKBP-EGFP-PARKIN and mito-tracker was then detected using a method similar to Example 5.1. The results are shown in Fig.70 When TSPO-Flag-FRB was used, the dimerization inducer promoted the localization of FKBP-EGFP-PARKIN to mitochondria. In contrast, when TSPO without FRB tag was used, no such promotion was observed, indicating that this promotion was not induced by the dimerization inducer alone, but was induced by the action of the FKBP-FRB system.

[0463] HeLa cells were transfected with FKBP-EGFP-PARKIN and Flag-TSPO or TSPO-Flag-FRB, and then treated with a dimerization inducer (1 μM) for 48 hours. Immunofluorescence was then performed using a method similar to that in Example 2.2, and it was observed that the dimerization inducer could not reduce the TOM20 level ( Fig.71 ). HeLa cells were transfected with mito-keima, FKBP-EGFP-PARKIN and Flag-TSPO or TSPO-Flag-FRB, and then treated with dimerization inducer (1 μM) for 48 hours. CCCP (5 μM) was used as a positive control. Then, the detection was performed using a method similar to Example 1.1, and it was observed that the dimerization inducer could not increase the mito-keima red signal ( Fig.72 ). The results showed that although PARKIN was successfully recruited to mitochondria by the FKBP-FRB system, mitophagy was not enhanced.

Claims

1. Compounds of formula (I): LCM-L-MTM (I) or a pharmaceutically acceptable salt thereof, in, MTM is the mitochondrial binding part; L is the joint part; LCM is the LC3 binding moiety.

2. The compound of claim 1, wherein The LCM moiety is a structure of the following formula (1) or a pharmaceutically acceptable salt thereof: in, Y is O or S; The C ring is selected from C 6-10 Aryl and five- to seven-membered heteroaryl, the aryl or heteroaryl is optionally substituted by one or more independently selected from R 1 The group substitution; L 01 is a bond, or is a C1-C6 hydrocarbon chain; R 01 , R 02 , R 03 , R 04 Each independently selected from H and R X2 ; R X1 and R X2 is independently selected at each occurrence from halogen, -NO2, -CN, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR 05 、-SR 05 、-NR 05 R 06 、-C(=O)OR 05 、-C(=O)NR 05 R 06 、-OC(=O)R 05 、-NC(=O)R 05 R 06 、-C(=O)R 05 、-S(=O)2OR 05 、-S(=O)2R 05 、-S(=O)2NR 05 R 06 、-OS(=O)2R 05 、-NS(=O)2R 05 R 06 、-S(=O)R 05 wherein the alkyl, alkenyl or alkynyl is optionally substituted by one or more selected from halogen, -NO2, -CN, -OH, -O(C 1-6 Alkyl), -O(C 3-6 Cycloalkyl), -O(C 1-4 Alkylene-C 3-6 Cycloalkyl), -O(three-membered to seven-membered heterocyclic group), -O(C 1-4 Alkylene)-(three-membered to seven-membered heterocyclic group), -SH, -S(C 1-6 Alkyl), -S(C 3-6 Cycloalkyl), -S(C 1-4 Alkylene-C 3-6 Cycloalkyl), -S(three-membered to seven-membered heterocyclic group), -S(C 1-4 Alkylene)-(three-membered to seven-membered heterocyclic group), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -NH(C 3-6 Cycloalkyl), -N(C 3-6 Cycloalkyl)2, -NH(C 1-4 Alkylene-C 3-6 Cycloalkyl), -N(C 1-4 Alkylene-C 3-6 Cycloalkyl)2, -NH(three-membered to seven-membered heterocyclic group), -N(three-membered to seven-membered heterocyclic group), -NH(C 1-4 Alkylene-three to seven-membered heterocyclic group), -N(C 1-4 alkylene-three to seven membered heterocyclic group)2, =O, -COOH and C 1-6 Substitution of alkyl groups; R 05 , R 06 Each occurrence is independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 Cycloalkyl-C 1-4 Alkyl, three-membered to seven-membered heterocyclic group, three-membered to seven-membered heterocyclic group-C 1-4 Alkyl, C 6-10 Aryl-C 1-4 wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl is optionally substituted by one or more selected from halogen, -NO2, -CN, C 1-6 Alkyl, -OH, -O(C 1-6 Alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 alkyl)2, -COOH, -C(=O)O(C 1-6 alkyl), -C(=O)NH(C 1-6 alkyl), -C(=O)N(C 1-6 Alkyl)2, -OC(=O)(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -C(=O)(C 1-6 alkyl) substituent.

3. The compound of claim 2, wherein The C ring is a five-membered to six-membered heteroaryl group, which is optionally substituted by 1, 2, 3, 4 or 5 groups each independently selected from R X1 Preferably, the C ring is a five-membered heteroaryl group containing 1 or 2 N atoms, optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R X1 In particular, the C ring is a benzene ring or a pyrrole; L 01 is a methylene or methine group, preferably 4. The compound of any one of claims 1 to 3, wherein Formula (1) has the structure of the following formula (2): in, R 1 Selected from halogen, -NO2, -CN and C 1-6 Alkyl, wherein the alkyl is optionally substituted with one or more substituents selected from halogen, -NO2 and -CN; R 2 , R 3 and R 4 is independently selected at each occurrence from halogen, -NO2, -CN, C 1-6 Alkyl, -OR 5 、-SR 5 、-NR 5 R 6 wherein the alkyl group is optionally substituted by one or more selected from halogen, -NO2, -CN, C 1-6 Alkyl, -OR 5 、-SR 5 、-NR 5 R 6 Substituents are substituted; R 5 and R 6 is independently selected at each occurrence from hydrogen and C 1-6 alkyl.

5. The compound of claim 4, wherein R 1 is selected from halogen, in particular I or Br.

6. The compound of any one of claims 4-5, wherein R 2 is selected from halogen, especially Br.

7. The compound of any one of claims 4 to 6, wherein R 3 Selected from halogen and -OR 5 , preferably -OR 5 , especially OH.

8. The compound of any one of claims 4 to 7, wherein R 4 Selected from halogen and -OR 5 , especially Br or OH.

9. The compound of any one of claims 4 to 7, wherein The structure of formula (1) or formula (2) is selected from:

10. The compound of any one of claims 1 to 8, wherein L is a structure of the following formula (i) or a pharmaceutically acceptable salt thereof: -Dy-(i) in, y is an integer greater than 1; and Each D is independently selected from the group consisting of: a bond, -CR L1 R L2 -, -O-, -S-, -S(=O)-, S(=O)2-, -NR L3 -、-S(=O)2NR L3 -、-S(=O)NR L3 -、-C(=O)NR L3 -、-NR L3 C(=O)NR L4 -、-NR L3 S(=O)2NR L4 -, -C(=O)-, -CR L1 =CR L2 -、-C≡C-、-SiR L1 R L2 -、-P(=O)R L1 -、-P(=O)OR L1 -, optionally through 0-6 R L1 and / or R L2 C 3-6 Cycloalkyl, optionally with 0-6 R L1 and / or R L2 A three- to seven-membered heterocyclic group substituted by a group, optionally substituted by 0-6 R L1 and / or R L2 The aryl group substituted by a group, optionally substituted by 0-6 R L1 and / or R L2 A heteroaryl group substituted with a group; wherein Each R L1 , R L2 , R L3 , R L4 and R L5 Each independently selected from: H, halogen, C 1-6 Alkyl, -O(C 1-6 Alkyl), -S(C 1-6 Alkyl), -NH(C 1-6 Alkyl), -N(C 1-6 alkyl)2, -OH, -NH2, -SH, halogen, -CN, -CF3, -CHF2, -CH2F and -NO2; preferably selected from H, halogen, C 1-6 Alkyl, -OH, -NH2, -SH, halogen, -CN, -CF3, -CHF2, -CH2F and -NO2; When y is greater than 1, R L1 or R L2 Each independently can be linked to another D group to form a further R L5 The cycloalkyl and / or heterocyclyl moieties are substituted by radicals.

11. The compound of claim 10, wherein y is 14; D is selected from -CR L1 R L2 -、-O-、-NR L3 -、-C(=O)NR L3 - and -C(=O)-; R L1 , R L2 and R L3 is H; In particular, the structure of L is 12. The compound of any one of claims 1 to 11, wherein The MTM moiety is a moiety capable of non-covalently interacting with mitochondria, preferably capable of non-covalently interacting with the mitochondrial outer membrane protein (OMM); Preferably, the MTM moiety is a moiety capable of non-covalently interacting with TOM20, TOM40, TOM70, TSPO, OMP25 or SAM50; More preferably, the MTM moiety is a moiety capable of non-covalently interacting with TSPO; Further preferably, the MTM moiety is selected from molecules that are affinity to TSPO, preferably 2-phenyl-1H-indole, in particular 13. A compound according to any one of claims 1 to 12, selected from 14. A pharmaceutical composition comprising a preventively or therapeutically effective amount of a compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

15. Use of the compound of any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 14 for promoting mitochondrial degradation; preferably, the degraded mitochondria are damaged or dysfunctional mitochondria; in particular, the damage includes but is not limited to depolarization damage.

16. Use of a compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 14, in the preparation of a medicament for treating a disease, disorder or condition selected from diseases associated with mitochondrial dysfunction.

17. The use of claim 16, wherein The disease, disorder or condition is neurodegenerative disease, myocardial disease, inflammation, cancer and aging; Preferably, the neurodegenerative disease is selected from Parkinson's disease (PD), Down syndrome (DS) and Alzheimer's disease (AD), in particular selected from Parkinson's disease and Down syndrome; Preferably, the myocardial disease is selected from heart failure and dilated cardiomyopathy (DCM); Preferably, the inflammation is selected from arthritis, inflammatory bowel disease, tendinitis, hepatitis, pancreatitis, vasculitis, contact dermatitis, psoriatic arthritis, ankylosing spondylitis and systemic lupus erythematosus (lupus); Preferably, the cancer is selected from: (a) a solid tumor or a blood-derived tumor selected from the following cancers: bladder cancer, endometrial cancer, squamous cell lung cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, esophageal cancer, gallbladder cancer, brain cancer, head and neck cancer, ovarian cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer, prostate cancer and skin cancer; (b) a hematopoietic tumor of the lymphoid system selected from the following: leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (c) a hematopoietic neoplasm of the myeloid lineage selected from the group consisting of acute and chronic myeloid leukemias, myelodysplastic syndromes and promyelocytic leukemias; (d) a tumor of mesenchymal origin selected from the group consisting of fibrosarcoma and rhabdomyosarcoma; (e) a tumor of the central and peripheral nervous system selected from the group consisting of astrocytoma, neuroblastoma, glioma and schwannoma; or (f) a melanoma, a seminoma, a teratoma, an osteosarcoma, a xeroderma pigmentosum, a keratoacanthoma, a follicular thyroid carcinoma or a Kaposi's sarcoma.

Images