Use of aromatic nitroethylenes

Abstract

The present application provides the use of aromatic nitroethylenes, in particular, the present application provides the use of a compound represented by formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof for preparing a medicament for modulating the dimer activity of nuclear receptor RXR or treating neurodegenerative diseases.

Description

Technical Field

[0001] This application relates to the pharmaceutical and biological fields. Specifically, this application relates to the use of aromatic nitroethylene compounds in modulating the activity of RXRα dimers, and their use in the preparation of medicaments for treating neurodegenerative diseases, particularly Alzheimer's disease. Background Technology

[0002] 1. Alzheimer's disease

[0003] Alzheimer's disease (AD) is a fatal neurodegenerative disease characterized by a decline in cognitive and memory abilities. With societal development and an accelerating aging population, the prevalence of AD is gradually increasing. Statistics show that over 50 million people aged 65 and over worldwide suffer from AD, and this number is projected to increase by 50% by 2020. my country is gradually entering an aging society, and currently has approximately 3 to 4 million AD patients. AD affects patients' lives in various ways, with symptoms primarily manifesting as a gradual loss of the brain's ability to store new information. This is usually caused by the death and dysfunction of first-order neurons involved in memory formation, as well as the death and dysfunction of nerve cells in other areas of the brain. AD has become the fourth leading cause of death among the elderly, rivaling cardiovascular disease, cancer, and cerebral thrombosis, seriously endangering their physical health and quality of life.

[0004] 1.1 AD Pathogenesis - Amyloid Cascade Hypothesis

[0005] Alzheimer's disease (AD) is currently believed to be a neurodegenerative disease, primarily caused by a combination of environmental, genetic, and social factors, and mainly manifests as damage to the nervous system. Several hypotheses have been proposed regarding the pathogenesis of AD, including the amyloid cascade hypothesis, the tau hypothesis, and the oxidative stress hypothesis. Among these, the amyloid cascade hypothesis has received the most attention, as it provides a robust theoretical framework for explaining the pathogenesis of AD.

[0006] Amyloid plaques are primarily formed by the extraneuronal aggregation of β-amyloid peptide (amyloid β, Aβ) to form amyloid filaments, which further aggregate to form plaques. Aβ is a protein peptide of 39–43 amino acid residues in length, produced in vivo from the metabolism of amyloid precursor protein (APP). APP is converted into Aβ under the action of β and γ secretases. 40 / 42 , Aβ 40 Aβ accounts for approximately 90% of the total. 42 It accounts for 10%, of which only 10% is Aβ. 42It readily forms dense, fibrous neuritis plaques characteristic of Alzheimer's disease (AD) and exhibits specific toxicity to the nervous system. In a normal human body, the amount of Aβ is constant; abnormalities occur when this balance is disrupted by various stimuli.

[0007] The amyloid cascade hypothesis posits that amyloid plaques are the most significant neurotoxic substance in Alzheimer's disease (AD), and that an imbalance between the production and clearance of its component, abelanoid (Aβ), is the cause of AD pathogenesis. Aβ aggregates cause a series of pathological changes in the brain tissue of patients, such as synaptic loss, inflammation, oxidative stress, and ultimately, neuronal degeneration. Current research indicates that Aβ is heavily deposited in the brains of AD patients. The carboxyl terminus of Aβ spontaneously aggregates to form nuclear factor, which forms a transparent Aβ fibril around its core. Through continuous β-folding, this ultimately forms Aβ filaments that can be observed under an electron microscope.

[0008] 1.2 Neurotoxicity of Aβ protein

[0009] While not the sole cause of Alzheimer's disease, Aβ protein plays a central role in its development. First, impaired clearance and degradation of Aβ protein lead to its deposition and plaque formation. Studies have shown that AD patients exhibit reduced phagocytic clearance of Aβ protein by monocytes and macrophages, resulting in ineffective and timely Aβ removal and triggering an immune response. Second, Aβ protein can promote neuronal apoptosis. Aβ can cause mitochondrial damage, oxidative stress, and calcium deficiency. 2+ Overload and decreased expression and activity of cyclooxygenase (COX) can lead to the release of cytochrome c and activation of cysteine ​​and aspartic proteases, initiating neuronal apoptosis. Simultaneously, mitochondrial dysfunction releases free radicals, resulting in severe oxidative stress in both AD patients and the brains of normal aging individuals. Furthermore, Aβ can activate glial cells, releasing cytokines and inflammatory mediators, generating an inflammatory response. As a stimulatory substance, Aβ accelerates neuronal death by activating microglia to produce cytokines and the nervous system's immune inflammatory response, leading to memory loss and cognitive impairment. Finally, Aβ protein is not only a crucial factor in the formation of neurofibrillary tangles (NFTs) but can also induce neuronal apoptosis through NFTs. The production and deposition of Aβ can have toxic effects on and damage mitochondria, causing calcium and other neuronal degradation. 2+ Overload activates calmodulin-dependent protein kinase II, leading to tau hyperphosphorylation, which prevents microtubules from assembling properly and causes neurofibrillary tangles.

[0010] 1.3 Clearance of Aβ

[0011] Due to the crucial role of Aβ in the pathological process of Alzheimer's disease (AD), the idea of ​​inhibiting AD development by reducing Aβ production or increasing Aβ clearance has been widely accepted. Since the metabolism of APP in vivo requires the participation of enzymes, these enzymes have become potential targets for AD treatment. Alpha-secretase participates in the formation of non-amyloid proteins while inhibiting the formation of amyloid proteins; therefore, enhancing the function of alpha-secretase appears to be a promising approach for treating AD. Beta-secretase initiates the amyloid metabolic pathway in APP, and the development of β-secretase inhibitors is challenging because this enzyme has many substrates and a broad substrate-binding domain. Gamma-secretase was the first target pursued in drug development. However, research has revealed that gamma-secretase is not a simple transmembrane protease; it comprises at least four proteins. Due to its structural complexity, obtaining high-resolution structural information of the active site is difficult, making it challenging as a drug target.

[0012] On the other hand, research shows that the mere presence of amyloid protein does not cause neurodegeneration; amyloid protein must aggregate to form plaques, and these Aβ plaques are the main cause of neurotoxicity. Therefore, another reasonable approach in anti-amyloid therapy is to increase Aβ clearance, thereby preventing the formation of amyloid plaques, reducing amyloid-related neurotoxicity, and ultimately treating AD. Studies have found that Aβ levels in AD patients and healthy individuals... 40 and Aβ 42 The average production rate was almost identical in the AD patient group. 42 The clearance rate was lower in the patient group than in the control group. The clearance rate was 5.3% per hour in the patient group, compared to 7.6% in the control group. These data suggest that increasing clearance is more important for the treatment of Alzheimer's disease.

[0013] 1.4 Aβ Clearance Mechanism

[0014] Apolipoprotein E (ApoE) is a plasma protein involved in cholesterol transport. Its two separate folded regions function to bind lipids in the blood and to cell surface receptors (such as LDL receptors). After hydrolysis by thrombin, the N-terminal region contains four helical regions, which are responsible for binding to cell surface receptors; the C-terminus is a highly helical α-helix region, which is involved in lipid binding. In the brain, ApoE is synthesized by astrocytes, oligodendrocytes, and activated microglia. ApoE is a major lipid transporter in the brain and plays an important role in the repair and regeneration of neuronal membranes. Furthermore, inflammation plays a crucial role in the pathogenesis of Alzheimer's disease (AD), and ApoE possesses anti-inflammatory activity.

[0015] The lipid transporter ABCA1, which transports cholesterol and phospholipids from the plasma membrane to apolipoproteins, is the rate-limiting step in the formation of high-density lipoprotein (HDL). ABCA1 is a member of the ABC (ATP-binding cassette transporter) superfamily. The ABCA1 gene encodes a 254 kDa transporter composed of 2261 amino acids. Structurally, the ABCA1 protein has two transmembrane portions and two nucleotide-binding regions. The nucleotide-binding sites bind ATP to provide energy for transmembrane transport. Each transmembrane portion has six transmembrane helices, and a highly hydrophobic regulatory region is located in the middle of the protein. Substrates such as free cholesterol and phospholipids are transported through this channel. ABCA1 is an integrated membrane protein that uses ATP as an energy source to promote the efflux of free cholesterol and phospholipids from the cell. ApoA-1, bound to the cell surface, binds to the free cholesterol and phospholipids transported by ABCA1, forming nascent HDL. In peripheral tissues, the primary recipient of cholesterol and phospholipids transported by ABCA1 is ApoA-1, while in the brain, the main recipient is ApoE. Experiments have shown that knocking out the ABCA1 gene in mice leads to a decrease in the ability of ApoE to receive lipids in the central nervous system, thereby exacerbating amyloid plaque deposition in the brains of AD mice. Conversely, selective overexpression of ABCA1 increases the degree of ApoE lipidation in the brain, thus preventing the formation of amyloid plaques in mice.

[0016] 2. Nuclear receptor RXRα

[0017] 2.1 Retinoid X receptor

[0018] Retinoid X receptors (RXRs) are an important class of nuclear receptor superfamily members, belonging to the non-steroidal receptor family. They are ligand-regulated transcriptional regulators involved in almost all physiological activities in the human body, including metabolism, growth, development, differentiation, death, and immunity. Abnormalities in their expression or function are closely related to many human diseases. RXRs are classified into three subtypes based on differences in amino acid sequence, response to natural and synthetic ligands, and ability to regulate the expression of downstream target genes: RXRα, RXRβ, and RXRγ. The distribution of each RXR subtype varies in different cells, tissues, and organs. RXRα is mainly expressed and distributed in the epidermis, intestine, kidney, and liver; while RXRβ is expressed and distributed in various tissues and organs; RXRγ is mainly expressed and distributed in brain and muscle tissue, with lower expression in adipose tissue. Among the three RXR subtypes, RXRα is the most important, widely distributed in various tissues of the organism, and performs vital functions.

[0019] 2.2 Structure of RXRα

[0020] Like other members of the nuclear receptor family, the nuclear receptor RXRα has six main functional domains. Starting from the N-terminus, these domains are as follows: a ligand-independent activation domain (AF-1), also known as the A / B region, whose function is related to a specific sequence in the promoter; next is the DNA-binding domain (DBD), also known as the C region, which is highly conserved and consists of two highly conserved zinc finger structures responsible for binding the nuclear receptor to DNA binding elements; following the DNA-binding domain is the hinge region, also known as the D region, connecting the DNA-binding domain to the ligand-binding domain; the ligand-binding domain (LBD), also known as the E region, consists of 12 α-helices and two β-sheets, with the 12th α-helix sequence being highly conserved and forming the core region of the transcriptional activation domain AF2. The α-helices and β-sheets in the LBD region are arranged in a helical sandwich structure. The ligand-binding pocket (LBP), a hidden, hydrophobic pocket located inside the center of this structure, provides a site for ligand binding. Under the influence of ligands, the conformation of the receptor changes, affecting the transcriptional activity of RXRα in forming homodimers or altering the intracellular localization of RXRα, thereby affecting various physiological processes of the cell; finally, there is the F domain, the function of which still needs to be studied.

[0021] 2.3 Functions of RXRα

[0022] The genotypic function of nuclear receptors primarily involves regulating target gene expression through direct interaction between the nuclear receptor and corresponding elements on DNA, or through interaction with transcription factors without direct DNA binding. Among the many members of the nuclear receptor superfamily, the RXR is a crucial and unique member, often referred to as the "Master Regulator." Many nuclear receptors exert their physiological functions by forming heterodimers with RXR. RXR exerts its function by regulating target gene transcription through either homodimer or heterodimer mechanisms. RXR can form heterodimers with peroxisome proliferator-activated receptor (PPAR), retinoic acid receptor (RAR), thyroid hormone receptor (TR), farnesoyl X receptor (FXR), vitamin D3 receptor (VDR), liver X receptor (LXR), pregnane X receptor (PXR), and Nur77 (nerve growth factor-induced gene B, NGFI-B). For heterodimers such as RXR / PPAR, RXR / Nur77, RXR / LXR, RXR / PXR, and RXR / FXR, the ligand of RXR alone is sufficient to activate the activity of the dimer; these heterodimers are called "permissive heterodimers". For RXR / RAR, RXR / VDR, and RXR / TR, the agonist of RXR must act simultaneously with the corresponding ligand that forms the heterodimer receptor to activate heterodimer activity. These heterodimers are called "non-permissive heterodimers".

[0023] Generally, RXRα and other nuclear receptors, when forming heterodimers, significantly enhance their DNA binding and transcriptional regulation. In the absence of ligands, some nuclear receptors bind to DNA and inhibit the transcription of target genes by interacting with transcriptional repressors. When transcriptionally activating ligands bind to nuclear receptors, they cause a conformational change in the receptor, releasing co-repressors, binding co-activators, and promoting transcription by the protein transcription apparatus.

[0024] 3. Small molecule ligands of RXRα

[0025] Retinoids are a class of natural or synthetic vitamin A derivatives that possess various biological effects, including regulating growth, differentiation, and apoptosis, primarily mediated by two receptor types: RXR and RAR. Both natural all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA) can activate RAR, but only 9-cis-RA can activate RXR. Crystallographic studies have revealed significant differences in the lower bound photobinding (LBP) of RAR and RXR, regardless of ligand binding. RAR has an "I"-type LBP, while RXR has an "L"-type LBP. Therefore, linear ATRA can only bind to RAR, while the L-type 9-cis-RA can bind to RXR.

[0026] The first synthetic RXRα ligand with high selectivity was reported in 1994. Based on extensive research of the TTNPB series of RAR agonist compounds, Ligand Pharmaceuticals designed and synthesized a novel selective RXR agonist, drawing on key factors in its molecular structure. Targretin, one of these agonists, has already been approved for marketing as a treatment for psoriasis, acne, Kaposi's sarcoma, and cutaneous T-cell lymphoma. Additionally, it is worth mentioning that Muccio and colleagues at the University of Alabama at Birmingham first reported the synthesis of the RXRα agonist 9cUAB. Currently, the National Cancer Institute is conducting clinical trials of 9cUAB for cancer prevention.

[0027] Aromatic nitroethylene compounds, represented by Z-10, are a class of ligands that we have discovered for the first time selectively bind to RXRα and modulate the activity of RXRα.

[0028] 4. Effects of RXRα ligands on AD

[0029] Currently, it is believed that the expression of ABCA1, which mediates the lipidation process of ApoE, is regulated by liver X receptor α / β (LXRs). LXRα / β are ligand-regulated transcription factors, members of the nuclear receptor superfamily, regulating the transcription of multiple genes, including ABCA1 and ApoE. As transcriptional regulators, LXRs need to bind to RXRs to form heterodimers LXR / RXR, which then bind to specific DNA elements (LXREs) on target genes, thereby regulating target genes at the transcriptional level. PPARγ can also form heterodimers with RXRs. Like the LXR / RXR dimer, the PPAR / RXR heterodimer is a permitted heterodimer, which can be activated and exert transcriptional activity in the presence of only the RXR ligand. Exploring drugs for treating Alzheimer's disease (AD) from the perspective of RXRα opens up new avenues for AD treatment. Summary of the Invention

[0030] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0031] As used herein, the term "stereoisomer" refers to isomers that have the same order of atoms or groups of atoms but different spatial arrangements. In this application, the term "stereoisomerism" is divided into conformational and configurational isomerism, and configurational isomerism is further divided into cis-trans and optical isomerism. Therefore, in this application, "stereoisomer" includes all possible optical isomers and diastereomers, and any combination thereof. When the compounds described in this application contain alkene double bonds, unless otherwise specified, they include cis and trans isomers, and any combination thereof.

[0032] As used herein, the term “pharmaceutically acceptable salt” means (1) a salt formed by an acidic functional group (e.g., -COOH, -OH, -SO3H, etc.) present in the compounds of the present invention with a suitable inorganic or organic cation (base), such as a salt formed by the compounds of the present invention with an alkali metal or alkaline earth metal, an ammonium salt of the compounds of the present invention, and a salt formed by the compounds of the present invention with a nitrogen-containing organic base; and (2) a salt formed by a basic functional group (e.g., -NH2, etc.) present in the compounds of the present invention with a suitable inorganic or organic anion (acid), such as a salt formed by the compounds of the present invention with an inorganic acid or an organic carboxylic acid.

[0033] Therefore, the "pharmaceutically acceptable salts" of the compounds of this invention include, but are not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, and N-benzyl-phenylethylamine. Salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts; hydrohalic acid salts, such as hydrofluoric acid salts, hydrochloride salts, hydrobromide salts, hydroiodide salts, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkyl sulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; aryl sulfonates, such as benzenesulfonates, p-benzenesulfonates, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts, such as glycine salts, trimethylglycine salts, arginine salts, ornithine salts, glutamate salts, aspartate salts, etc.

[0034] As used in this article, the term "C" 1-6 "Alkyl" refers to a straight-chain or branched alkyl group containing 1-6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc. 1-6 Preferred examples of alkyl groups include C 1-5 Alkyl, C 1-4 Alkyl, C 1-3 Alkyl, C 3-6 Alkyl and C 3-5 Alkyl groups, etc.

[0035] As used in this article, the term "C" 1-6 "Alkoxy" refers to a group with a C 1-6 Alkyl-O- groups, wherein "C" 1-6 The definition of "alkyl" is as described above. Examples include methoxy, ethoxy, n-propoxy, and isopropoxy. C 1-6 Preferred examples of alkoxy groups include C 1-5 Alkoxy, C 1-4 Alkoxy, C 1-3 Alkoxy, C 3-6 Alkoxy and C 3-5Alkyl groups, etc.

[0036] As used in this article, the term "C" 1-6 "Alkylamine group" refers to a group with C 1-6 alkyl-NH- groups, wherein "C 1-6 The definition of "alkyl" is as described above. Examples include methylamino, ethylamino, n-propylamino, and isopropylamino. C 1-6 Preferred examples of alkylamine groups include C 1-5 alkylamine group, C 1-4 alkylamine group, C 1-3 alkylamine group, C 3-6 alkylamine group and C 3-5 Alkylamine group, etc.

[0037] As used in this article, the term "C" 1-6 "Alkylthio" refers to a group with a C group. 1-6 Alkyl-S-structure groups, wherein "C" 1-6 The definition of "alkyl" is as described above. Examples include methylthio and ethylthio. C 1-6 Preferred examples of alkylthio groups include C 1-5 Alkylthio, C 1-4 Alkylthio, C 1-3 Alkylthio, C 3-6 alkylthio and C 3-5 Alkylthio groups, etc.

[0038] As used herein, the term "halogen" includes, for example, fluorine, chlorine, bromine, and iodine atoms.

[0039] As used herein, the term "5-6 heteroaryl" refers to an aromatic group containing 5-6 ring atoms, at least one of which is a heteroatom. Specific examples of "5-6 membered heteroaryl groups" include, but are not limited to, furanyl, thiopheneyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, pyrimidinyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, and 1,2,4,5-tetraazinyl.

[0040] The term “regulation” as used in this article includes positive regulation (e.g., maintenance, upregulation, or activation) and negative regulation (e.g., downregulation or inhibition).

[0041] The pCMV-myc-RXRα plasmid mentioned in this article refers to the pCMV plasmid that encodes the RXRα protein with the myc tag.

[0042] The pCMV-myc-RARα plasmid mentioned in this article refers to the pCMV plasmid that encodes the RARα protein with the myc tag.

[0043] The pCMV-myc-PPARγ plasmid mentioned in this article refers to the pCMV plasmid that encodes the PPARγ protein with the myc tag.

[0044] The pCMV-myc-LXRα plasmid mentioned in this article refers to the pCMV plasmid that encodes the LXRα protein with the myc tag.

[0045] The pBIND-RXRαLBD plasmid mentioned in this article refers to the pBIND plasmid encoding the Gal4 / DBD-RXRα / LBD fusion protein.

[0046] The pBIND-RARαLBD plasmid mentioned in this article refers to the pBIND plasmid encoding the Gal4 / DBD-RARα / LBD fusion protein.

[0047] The pBIND-ERαLBD mentioned in this article refers to the pBIND plasmid encoding the Gal4 / DBD-ERα / LBD fusion protein.

[0048] The pGL6-TA-RXRE-luciferase plasmid mentioned in this article refers to a reporter gene plasmid that responds to RXR / RXR homodimers and is used to detect the transcriptional activity of RXR / RXR homodimers, where RXRE refers to the DNA binding element of RXR / RXR homodimers.

[0049] The pGL6-TA-RARE-luciferase plasmid mentioned in this article refers to a reporter gene plasmid that responds to RXR / RAR heterodimers and is used to detect the transcriptional activity of RXR / RAR heterodimers, where RARE refers to the DNA binding element of RXR / RAR heterodimers.

[0050] The pGL6-TA-PPRE-luciferase plasmid mentioned in this article refers to a reporter gene plasmid that responds to the RXR / PPAR heterodimer, where PPRE refers to the DNA binding element of the RXR / PPAR heterodimer.

[0051] The pGL6-TA-LXRE-luciferase plasmid mentioned in this article refers to a reporter gene plasmid that responds to the RXR / LXR heterodimer, where LXRE refers to the DNA binding element of the RXR / LXR heterodimer.

[0052] The pG5-luciferase plasmid mentioned in this article refers to the reporter gene plasmid of the corresponding Gal4 / DBD fusion protein.

[0053] The renilla-luciferase plasmid mentioned in this article refers to the sea cucumber luciferase plasmid, which is used as an internal control in reporter gene experiments.

[0054] The inventors of this application have surprisingly discovered, during their research on the biological function of the nuclear receptor RXRα and optimization of its novel ligand Z-10, that modified ligands (especially those modified with alkyl and alkoxy groups at the 2-position of the nitrovinyl group) can significantly enhance the ability of compounds to activate the transcriptional activity of RXRα homodimers and heterodimers (e.g., RXRα / RARα, RXRα / PPARγ, or RXRα / LXRα). In some embodiments of this application, the ligand can regulate the physiological function of the corresponding nuclear receptor by modulating the transcriptional activity of the dimer, thereby enabling its use in the treatment of related diseases. This invention is based on the above findings.

[0055] Therefore, in one aspect, this application provides the use of the compound of formula (I), its stereoisomers, or pharmaceutically acceptable salts for regulating the activity (e.g., transcriptional activity) of the dimer of the nuclear receptor RXR, or for preparing a medicament for regulating the activity (e.g., transcriptional activity) of the dimer of the nuclear receptor RXR.

[0056]

[0057] Among them, R1, R2, and R3 are each independently selected from hydrogen, halogen, hydroxyl, amino, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio;

[0058] R4 and R5 form a benzene ring or a 5-6 membered heteroaromatic ring, wherein the benzene ring or the 5-6 membered heteroaromatic ring is optionally substituted by one or more (e.g., 1, 2, 3, or 4) substituents selected from the following: halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio group.

[0059] In some preferred embodiments, R4 and R5 form a benzene ring.

[0060] In some preferred embodiments, the structure of the compound is shown in formula (II).

[0061]

[0062] R1 and R3 are each independently selected from hydrogen, halogen, hydroxyl, amino, and C. 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 alkylamine group and C 1-4 Alkylthio group, and only one of R1 and R3 is hydrogen;

[0063] R6 is selected from hydrogen, halogen, hydroxyl, amino, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 alkylamine group and C 1-4 Alkylthio group.

[0064] In some preferred embodiments, R1 is selected from hydroxyl, C 1-4 Alkyl and C 1-4 Alkoxy group; preferably C 1-4 Alkyl or C 1-4 Alkoxy;

[0065] R3 is hydrogen;

[0066] R6 is selected from hydrogen, halogen, hydroxyl, amino, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 alkylamine group and C 1-4 Alkylthio group.

[0067] In some preferred embodiments, R1 is hydrogen;

[0068] R3 is selected from hydroxyl group, C 1-4 Alkyl and C 1-4 Alkoxy group; preferably C 1-4 Alkyl or C 1-4 Alkoxy;

[0069] R6 is selected from hydrogen, halogen, hydroxyl, amino, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 alkylamine group and C 1-4 Alkylthio group.

[0070] In some preferred embodiments, the compound is selected from:

[0071]

[0072] In some preferred embodiments, the dimer of the nuclear receptor RXR is a homodimer or heterodimer of the nuclear receptor RXR.

[0073] In some preferred embodiments, the heterodimer of the RXR is selected from the dimer formed by RXR with RAR, PPAR or LXR.

[0074] In some preferred embodiments, the heterodimer of the RXR is selected from RXRα / RARα, RXRα / PPARγ, and RXRα / LXRα.

[0075] In embodiments of the present invention, the compound can be used as a ligand for RXR (e.g., RXRα) to regulate the activity of RXR dimers (e.g., transcriptional activity).

[0076] In another aspect, this application provides the use of the compound of formula (I), its stereoisomers, or pharmaceutically acceptable salts in the preparation of medicaments for reducing Aβ plaques.

[0077]

[0078] Among them, R1, R2, and R3 are each independently selected from hydrogen, halogen, hydroxyl, amino, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio;

[0079] R4 and R5 form a benzene ring or a 5-6 membered heteroaromatic ring, wherein the benzene ring or the 5-6 membered heteroaromatic ring is optionally substituted by one or more (e.g., 1, 2, 3, or 4) substituents selected from the following: halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio group.

[0080] In some preferred embodiments, the compound of formula (I) is as defined above.

[0081] In another aspect, this application provides the use of the compound of formula (I), its stereoisomers, or pharmaceutically acceptable salts in the preparation of medicaments for treating neurodegenerative diseases.

[0082]

[0083] Among them, R1, R2, and R3 are each independently selected from hydrogen, halogen, hydroxyl, amino, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio;

[0084] R4 and R5 form a benzene ring or a 5-6 membered heteroaromatic ring, wherein the benzene ring or the 5-6 membered heteroaromatic ring is optionally substituted by one or more (e.g., 1, 2, 3, or 4) substituents selected from the following: halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio group.

[0085] In some preferred embodiments, the compound of formula (I) is as defined above.

[0086] In some preferred embodiments, the neurodegenerative disease is Alzheimer's disease.

[0087] In another aspect, this application provides a combination product (e.g., a pharmaceutical composition) comprising ligand L and a compound of formula (I), its stereoisomer, or a pharmaceutically acceptable salt, wherein,

[0088] The ligand L is a ligand of a nuclear receptor that forms a dimer with the nuclear receptor RXR;

[0089] The compound of formula (I) is shown below:

[0090]

[0091] Among them, R1, R2, and R3 are each independently selected from hydrogen, halogen, hydroxyl, amino, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio;

[0092] R4 and R5 form a benzene ring or a 5-6 membered heteroaromatic ring, wherein the benzene ring or the 5-6 membered heteroaromatic ring is optionally substituted by one or more (e.g., 1, 2, 3, or 4) substituents selected from the following: halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio group.

[0093] In some preferred embodiments, the compound of formula (I) is as defined above.

[0094] In some preferred embodiments, the RXR dimer is a homodimer or heterodimer of RXR.

[0095] In some preferred embodiments, the heterodimer of the RXR is selected from the dimer formed by RXR with RAR, PPAR or LXR.

[0096] In some preferred embodiments, the heterodimer of the RXR is selected from RXRα / RARα, RXRα / PPARγ, and RXRα / LXRα.

[0097] In some preferred embodiments, the ligand L is selected from ligands of RARα (e.g., all-trans retinoic acid ATRA), ligands of PPARγ (e.g., rosiglitazone), and ligands of LXRα (e.g., T0901317).

[0098] In another aspect, this application provides the use of the combined product in regulating the activity of RXR dimers (e.g., transcriptional activity), or in the preparation of medicaments for regulating the activity of RXR dimers (e.g., transcriptional activity).

[0099] In another aspect, this application provides the use of the combined product in the preparation of a medicament for treating neurodegenerative diseases.

[0100] In some preferred embodiments, the neurodegenerative disease is Alzheimer's disease.

[0101] In another aspect, this application provides a method for regulating RXR dimer activity (e.g., transcriptional activity), which includes the step of contacting cells with an effective amount of the aforementioned compound or combination product.

[0102] In some preferred embodiments, the RXR dimer is a homodimer or heterodimer of RXR.

[0103] In some preferred embodiments, the heterodimer of the RXR is selected from the dimer formed by RXR with RAR, PPAR or LXR.

[0104] In some preferred embodiments, the heterodimer of the RXR is selected from RXRα / RARα, RXRα / PPARγ, and RXRα / LXRα.

[0105] In some preferred embodiments, the ligand L is selected from ligands of RARα (e.g., ATRA), ligands of PPARγ (e.g., rosiglitazone), and ligands of LXRα (e.g., T0901317).

[0106] In some preferred embodiments, the cells are cell lines or cells derived from the subject.

[0107] In some preferred embodiments, the cells are derived from the subject's brain tissue (e.g., hippocampus or cerebral cortex).

[0108] In another aspect, this application provides a method for treating neurodegenerative diseases, comprising the step of administering an effective amount of the compound or combination product as described above to a subject in need of such treatment.

[0109] In some preferred embodiments, the neurodegenerative disease is Alzheimer's disease.

[0110] In another aspect, this application provides the following compounds, their stereoisomers, or pharmaceutically acceptable salts,

[0111]

[0112] In another aspect, this application provides a method for enhancing the activity (e.g., transcriptional activity) of a compound of formula (III), its stereoisomer, or a pharmaceutically acceptable salt activating RXR or its dimer, comprising modifying the R1' position in formula (III) to C 1-6 Alkyl or C 1-6 The alkoxy step,

[0113]

[0114] R1' is selected from hydrogen, halogen, hydroxyl and amino groups;

[0115] R3 is selected from hydrogen, halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio;

[0116] R4 and R5 form a benzene ring or a 5-6 membered heteroaromatic ring, wherein the benzene ring or the 5-6 membered heteroaromatic ring is optionally substituted by one or more (e.g., 1, 2, 3, or 4) substituents selected from the following: halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio group.

[0117] In some preferred embodiments, R4 and R5 form a benzene ring.

[0118] In some preferred embodiments, the structure of the compound is as shown in formula (III)-1.

[0119]

[0120] R1' is selected from hydrogen, halogen, hydroxyl and amino groups;

[0121] R6 is selected from hydrogen, halogen, hydroxyl, amino, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 alkylamine group and C 1-4 Alkylthio group.

[0122] In some preferred embodiments, R1' is hydrogen;

[0123] R6 is selected from hydrogen, halogen, hydroxyl, amino, C 1-4 Alkyl, C1-4 Alkoxy, C 1-4 alkylamine group and C 1-4 Alkylthio group.

[0124] In some preferred embodiments, the compound is

[0125] In some preferred embodiments, the dimer of RXR is a homodimer or heterodimer of RXR.

[0126] In some preferred embodiments, the heterodimer of the RXR is selected from the dimer formed by RXR with RAR, PPAR or LXR.

[0127] In some preferred embodiments, the heterodimer of the RXR is selected from RXRα / RARα, RXRα / PPARγ, and RXRα / LXRα.

[0128] In another aspect, this application provides a method for downregulating the transcriptional activity of RXR or its dimer by a compound of formula (IV), its stereoisomer, or a pharmaceutically acceptable salt, comprising modifying the R3' position of formula (IV) to C 1-6 Alkyl or C 1-6 The alkoxy step,

[0129]

[0130] R1 is selected from hydrogen, halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio;

[0131] R3' is selected from hydrogen, halogen, hydroxyl, and amino;

[0132] R4 and R5 form a benzene ring or a 5-6 membered heteroaromatic ring, wherein the benzene ring or the 5-6 membered heteroaromatic ring is optionally substituted by one or more (e.g., 1, 2, 3, or 4) substituents selected from the following: halogen, hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group and C 1-6 Alkylthio group.

[0133] In some preferred embodiments, R4 and R5 form a benzene ring.

[0134] In some preferred embodiments, the structure of the compound is shown in formula (IV)-1.

[0135]

[0136] R3' is selected from hydrogen, halogen, hydroxyl and amino groups;

[0137] R6 is selected from hydrogen, halogen, hydroxyl, amino, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 alkylamine group and C 1-4 Alkylthio group.

[0138] In some preferred embodiments, R3' is hydrogen;

[0139] R6 is selected from hydrogen, halogen, hydroxyl, amino, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 alkylamine group and C 1-4 Alkylthio group.

[0140] In some preferred embodiments, the compound is

[0141] In some preferred embodiments, the dimer of RXR is a homodimer or heterodimer of RXR.

[0142] In some preferred embodiments, the heterodimer of the RXR is selected from the dimer formed by RXR with RAR, PPAR or LXR.

[0143] In some preferred embodiments, the heterodimer of the RXR is selected from RXRα / RARα, RXRα / PPARγ, and RXRα / LXRα. Attached Figure Description

[0144] Figure 1 This application shows the structure of the nitroethylene compounds described herein.

[0145] Figure 2 A shows the activation of Gal4 / DBD-RXRα / LBD transcriptional activity by Z-10 derivatives modified at positions 2 and 4, as determined by yeast one-hybrid assay. ＊＊＊ p<0.001, n≥3.

[0146] Figure 2 B shows the activation of Gal4 / DBD-RARα / LBD transcriptional activity by the Z-10 derivatives modified at positions 2 and 4, as determined by yeast one-hybrid assay.

[0147] Figure 2 C shows the activation of Gal4 / DBD-ERα / LBD transcriptional activity by the Z-10 derivatives modified at positions 2 and 4, as determined by yeast one-hybrid assay.

[0148] Figure 2D shows the activation of RXRα homodimer transcriptional activity by Z-10 derivatives modified at positions 2 and 4, as measured by reporter gene sequencing. ＊＊＊ p<0.001, n≥3.

[0149] Figure 2 E shows the activation of RXRα / RARα heterodimer transcriptional activity by Z-10 derivatives modified at positions 2 and 4, as measured by reporter gene sequencing. ＊＊＊ p<0.001, n≥3.

[0150] Figure 2 F shows the activation of RXRα / PPARγ heterodimer transcriptional activity by Z-10 derivatives modified at positions 2 and 4, as measured by reporter gene sequencing. ＊＊ p<0.01, ＊＊＊ p<0.001, n≥3.

[0151] Figure 2 G shows the activation of RXRα / LXRα heterodimer transcriptional activity by Z-10 derivatives modified at positions 2 and 4, as measured by reporter gene sequencing. ＊ p<0.05, ＊＊ p<0.01, ＊＊＊ p<0.001, n≥3.

[0152] Figure 3 A shows the binding of Z-10 to RXRα protein as determined by fluorescence titration; the illustrated concentration of Z-10 was gradually added to purified RXRα / LBD (5 μM) protein, and the emission spectrum of RXRα / LBD protein excited by 280 nm light from 300 to 450 nm was detected.

[0153] Figure 3 B shows the binding of Z-10 to RXRα protein as determined by fluorescence titration; among which... Figure 3 The experimental data of A were combined with Origin software and the formula "y = C". ＊ (x+5+K d ) / 2-C ＊ sqrt(x+5+K d ) 2 -4 ＊ 5 ＊ The K value of the combination of Z-10 and RXRα / LBD is calculated by x) / 2”. d value.

[0154] Figure 3C shows the binding of the Z-10 derivatives modified at positions 2 and 4 to RXRα protein as determined by fluorescence titration; the illustrated concentration of compound Z was gradually added to purified RXRα / LBD (5 μM) protein, and the emission spectrum of RXRα protein excited by 280 nm light at 300-450 nm was detected. The experimental data were then analyzed using Origin software and the formula "y = C". ＊ (x+5+K d ) / 2-C ＊ sqrt(x+5+K d ) 2 -4 ＊ 5 * The KZ binding to RXRα was calculated using x) / 2”. d value.

[0155] Figure 3 D shows the activation of RXRα transcriptional activity by the 4-position modified Z-10 derivative as determined by yeast one-hybrid assay.

[0156] Figure 4 A shows the activation of Gal4 / DBD-RXRα / LBD transcriptional activity by the 2-alkoxy modified Z-10 derivative, as determined by yeast one-hybrid assay. ＊＊＊ p<0.001, n≥3.

[0157] Figure 4 B shows the activation of Gal4 / DBD-RARα / LBD transcriptional activity by the 2-alkoxy modified Z-10 derivative, as determined by yeast one-hybrid assay.

[0158] Figure 4 C shows the activation of RXRα homodimer transcriptional activity by the 2-alkoxy modified Z-10 derivative, as measured by reporter gene sequencing. ＊＊＊ p<0.001, n≥3.

[0159] Figure 4 D shows the activation of RXRα homodimer transcriptional activity by the 2-alkoxy modified Z-10 derivative, as measured by reporter gene sequencing. ＊＊ p<0.01, n≥3.

[0160] Figure 4 E shows the activation of RXRα / RARα heterodimer transcriptional activity by the 2-alkoxy modified Z-10 derivative, as measured by reporter gene sequencing. ＊ p<0.05, ＊＊＊ p<0.001, n≥3.

[0161] Figure 5A shows the activation of the transcriptional activity of the RXRα / LXRα heterodimer by the Z-10 derivative modified at the 2-alkoxy position, as determined by reporter gene sequencing. ＊ p<0.05, n≥3.

[0162] Figure 5 B shows the activation of the transcriptional activity of the RXRα / PPARγ heterodimer by the Z-10 derivative with alkoxy group modification at position 2, as measured by reporter gene sequencing. ＊＊ p<0.01, n≥3.

[0163] Figure 5 C shows the activation of the transcriptional activity of the RXRα / LXRα heterodimer by the Z-10 derivative modified at the 2-alkoxy position, as determined by reporter gene sequencing. ＊＊ p<0.01, ＊＊＊ p<0.001, n≥3.

[0164] Figure 5 D shows the activation of RXRα / PPARγ heterodimer transcriptional activity by the 2-alkoxy modified Z-10 derivative, as measured by reporter gene sequencing. ＊＊＊ p<0.001, n≥3.

[0165] Figure 5 E shows the binding of Z-36 to RXRα protein as determined by fluorescence titration; the illustrated concentration of Z-36 was gradually added to the purified RXRα / LBD protein, and the emission spectrum of RXRα / LBD protein excited by 280nm light from 300-450nm was detected.

[0166] Figure 5 F shows the binding of Z-36 to RXRα protein as determined by fluorescent titration; where Figure 5 The experimental data of E were analyzed using Origin software and the formula "y = C". ＊ (x+5+K d ) / 2-C ＊ sqrt(x+5+K d ) 2 -4 ＊ 5 ＊ The K value of the combination of Z-10 and RXRα / LBD is calculated by x) / 2”. d value.

[0167] Figure 6 This study demonstrates the effects of Z-10 and Z-36 on Aβ plaques in the hippocampus, as measured in an AD female mouse model experiment. APP / PS1 transgenic female mice were administered corn oil (Vehicle), Z-10, and Z-36 by gavage at 40 mg / kg / day for 15 days. Hippocampal samples were then removed, stained with Aβ antibody for immunofluorescence, and the number of Aβ plaques was counted.＊ p<0.05, ＊＊＊ p<0.001, n≥3.

[0168] Figure 7 This study demonstrates the effects of Z-10 and Z-36 on Aβ plaques in the hippocampus, as measured in an AD male rat model. APP / PS1 transgenic male rats were administered corn oil (Vehicle), Z-10, and Z-36 by gavage for 15 days at a dose of 40 mg / kg / day. Hippocampal samples were then removed, stained with Aβ antibody for immunofluorescence, and the number of Aβ plaques was counted. ＊ p<0.05, ＊＊ p<0.01, ＊＊＊ p<0.001, n=5.

[0169] Figure 8 This study demonstrates the effects of Z-10 and Z-36 on Aβ plaques in the cerebral cortex, as measured in an AD female mouse model experiment. APP / PS1 transgenic female mice were administered corn oil (Vehicle), Z-10, and Z-36 by gavage at 40 mg / kg / day for 15 days. Cerebral cortex samples were then extracted, stained with Aβ antibody for immunofluorescence, and the number of Aβ plaques was counted. ＊ p<0.05, ＊＊ p<0.01, ＊＊＊ p<0.001, n=5.

[0170] Figure 9 This study demonstrates the effects of Z-10 and Z-36 on Aβ plaques in the cerebral cortex, as measured in an AD male rat model. APP / PS1 transgenic male rats were administered corn oil (Vehicle), Z-10, and Z-36 by gavage at 40 mg / kg / day for 15 days. Cerebral cortex samples were then extracted, stained with Aβ antibody using immunofluorescence, and the number of Aβ plaques was counted. ＊ p<0.05, ＊＊ p<0.01, ＊＊＊ p<0.001, n=5.

[0171] Figure 10 This study demonstrates the induction effects of Z-10 and Z-36 on ABCA1 and ABCG1 proteins in the hippocampus of AD model mice, as measured by Western blotting. AD model mice were administered corn oil (Vehicle), Z-10, and Z-36 by gavage for 15 days (40 mg / kg / day). Hippocampal tissue was then extracted for Western blotting to detect the expression of ABCA1, ABCG1, and apoE, and the immunoblot bands were analyzed using grayscale analysis. ＊ p<0.05, n≥3.

[0172] Figure 11 The compound Z-46 is shown 1HNMR spectrum.

[0173] Figure 12 The compound Z-46 is shown 13 CNMR spectrum.

[0174] Figure 13 The mass spectrum of compound Z-46 is shown.

[0175] Figure 14 The compound Z-49 is shown. 1 HNMR spectrum.

[0176] Figure 15 The compound Z-49 is shown. 13 CNMR spectrum.

[0177] Figure 16 The mass spectrum of compound Z-49 is shown.

[0178] Figure 17 The display of compound Z-52 1 HNMR spectrum.

[0179] Figure 18 The display of compound Z-52 13 CNMR spectrum.

[0180] Figure 19 The mass spectrum of compound Z-52 is shown.

[0181] Figure 20 The display of compound Z-53 1 HNMR spectrum.

[0182] Figure 21 The display of compound Z-53 13 CNMR spectrum.

[0183] Figure 22 The mass spectrum of compound Z-53 is shown. Detailed Implementation

[0184] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0185] I. Materials and Methods

[0186] 1. Experimental Materials

[0187] 1.1 Cell lines, strains, and compounds

[0188] 1.1.1 Cell lines

[0189] The African green monkey kidney cell line Cos-7 and the human kidney epithelial cell line HEK293T were derived from the American Type Culture Collection (ATCC) in the United States.

[0190] 1.1.2 Cloning host bacteria

[0191] Top 10 E. coli samples, purchased from Beyotime.

[0192] 1.1.3 Competent cells expressing proteins

[0193] Rosetta-gami(DE3)pLysS competent cells were purchased from Beyotime.

[0194] 1.1.3 Compounds

[0195] The Z-series compounds were obtained from the compound library of the Cancer Center of the School of Pharmacy, Xiamen University.

[0196] 1.2 Plasmids

[0197] The following plasmids were constructed and preserved in our laboratory: pCMV-myc-RXRα, pCMV-myc-RARα, pCMV-myc-PPARγ, pCMV-myc-LXRα, pBIND-RXRαLBD, pBIND-RARαLBD, pBIND-ERαLBD, pGL6-TA-RXRE-luciferase reporter gene plasmid, pGL6-TA-RARE-luciferase reporter gene plasmid, pGL6-TA-PPRE-luciferase, pG5-luciferase reporter gene plasmid, and pGL6-TA-LXRE-luciferase reporter gene plasmid.

[0198] 1.3 Laboratory Animals

[0199] The B6C3-Tg(APPswe,PSEN1dE9)85Dbo / J mouse, also known as the APP / PS1 mouse, was donated by Professor Xu Huaxi's laboratory at Xiamen University. It expresses a fusion of the mutant human presenilin (DeltaE9) and human-mouse amyloid precursor protein (APPswe), both of which are expressed by a mouse prion protein promoter. The DeltaE9 mutation in the human presenilin gene is caused by the deletion of the ninth exon, a mutation that leads to early-onset Alzheimer's disease. Researchers have reported that this strain of mice, aged 6-7 months, forms β-amyloid protein deposits in their brains, which can be used to establish Alzheimer's disease models.

[0200] 1.4 Main Reagents

[0201]

[0202]

[0203] 1.5 Main Instruments

[0204]

[0205]

[0206] 1.6 Main Solution

[0207] 1.6.1 PBS buffer:

[0208]

[0209] Bring the volume of deionized water to 1000 ml.

[0210] 1.6.2 Tissue lysis fluid

[0211] Lysis buffer I:

[0212]

[0213]

[0214] 1.6.3 Western blot related solutions

[0215] 1.5 mol / L Tris-HCl pH 8.8:

[0216] Tris(MW121.14) 181.72g

[0217] 800ml of ultrapure water

[0218] After dissolving, adjust the pH to 8.8 with concentrated hydrochloric acid, and finally bring the volume to 1000ml with ultrapure water. Store at room temperature.

[0219] 1.0 mol / L Tris-HCl pH 6.8

[0220] Tris(MW121.14) 121.1g

[0221] 800ml of ultrapure water

[0222] After dissolving, adjust the pH to 6.8 with concentrated hydrochloric acid, and finally bring the volume to 1000ml with ultrapure water. Store at room temperature.

[0223] Note: The solution should be cooled to room temperature before adjusting the pH value, because the pH value of Tris solution varies greatly with temperature. For every 1°C increase in temperature, the pH value of the solution decreases by approximately 0.03 units.

[0224] 10% SDS:

[0225] Dissolve 10g of electrophoretic grade SDS in 90ml of water, heat to 68℃ and stir until dissolved. Make up to 100mL. Store at room temperature.

[0226] 10% AP ammonium persulfate:

[0227] Dissolve 1g of ammonium persulfate in 10mL of deionized water by stirring, and store at 4℃.

[0228] 10% separating gel (10ml):

[0229]

[0230]

[0231] Add the above solutions to the small beakers in sequence, quickly, and after thorough mixing, quickly add them to the gel tank. Finally, add about 1 ml of anhydrous ethanol to the gel solution for sealing. It will solidify in 30-40 minutes at room temperature.

[0232] 5% stacking gel (4ml):

[0233]

[0234] After the separating gel solidifies, pour off the water on top and thoroughly blot it dry with filter paper. Prepare the gel according to the procedure, adding the gel solution completely and then quickly inserting a comb. It will solidify in 30–45 minutes at room temperature; simply remove the comb gently. If the prepared gel is not used immediately, it can be stored in a 4°C refrigerator.

[0235] 4×SDS gel loading buffer:

[0236]

[0237] Electrophoresis buffer:

[0238] Tris 3.03g

[0239] Glycine 18.77g

[0240] SDS 1g

[0241] Make up to 1000ml with deionized water.

[0242] Electroporation buffer:

[0243] Tris 3.03g

[0244] Glycine 14.42g

[0245] 10% methanol (add immediately before use)

[0246] Make up to 1000ml with deionized water.

[0247] TBST:

[0248] Tris 1.21g

[0249] NaCl 8.77g

[0250] Tween-20 1ml

[0251] Add deionized water to 1000ml and adjust the pH to 7.6.

[0252] Sealing fluid: 5% skim milk is dissolved in TBST solution.

[0253] Developer:

[0254] X-ray film developing powder (Canned Dragon brand) 1 pack

[0255] Add deionized water to a total volume of 3.785L.

[0256] Fixing solution:

[0257] X-ray film fixer powder (Canned Dragon brand) 1 pack

[0258] Add deionized water to a total volume of 3.785L.

[0259] 1.6.4 Solutions related to bacterial culture and gene cloning

[0260] LB solid medium:

[0261]

[0262] Add deionized water to 1000ml, autoclave at 121℃ for 30 minutes, then pour into a 10cm diameter container in a clean bench. 2 Plate the medium and store at 4°C after solidification. If using LB liquid medium, do not add agar.

[0263] TSS solution (competent bacterial culture medium):

[0264]

[0265]

[0266] Add LB (pH 6.5) to a final volume of 100 ml, filter to sterilize, and store at 4°C.

[0267] antibiotic:

[0268] Ampicillin solution: Prepare an ampicillin solution with a final concentration of 100 mg / ml using deionized water, filter through a 0.22 μm filter membrane, dispense into individual vials, and store at -20°C. Add the solution to the required concentration when using.

[0269] Kanamycin solution: Prepare a kanamycin solution with a final concentration of 50 mg / ml using deionized water. Filter the solution through a 0.22 μm filter membrane, dispense into individual vials, and store at -20°C. Add the solution to the required concentration when using.

[0270] Conversion enhancement solution (5×KCM):

[0271]

[0272] After autoclaving, dispense into smaller portions and store at -20°C for later use.

[0273] 1% agarose gel:

[0274] Weigh 0.5g of electrophoresis-grade agarose into an Erlenmeyer flask, add 50ml of deionized water, and heat the solution to boiling using a microwave oven. Repeat once. After the solution has cooled slightly, add the required amount of gel red. Then, slowly pour the gel solution into the gelation tank and allow it to solidify completely at room temperature. If not used immediately, it can be sealed with plastic wrap and stored at 4 degrees Celsius for later use.

[0275] DNA electrophoresis buffer 50×TAE:

[0276] Tris 242g

[0277] Na2EDTA·2H2O 37.2g

[0278] Dissolve the contents in approximately 800ml of water first, then add 57.1ml of acetic acid and stir thoroughly to bring the volume to a final volume.

[0279] 1.6.5 Protein Extraction Related Solutions

[0280] Buffer A:

[0281] Sodium phosphate 250mM

[0282] Sodium chloride 1500mM

[0283] Imidazole 100mM

[0284] Dissolve the substance in approximately 900ml of water, stir thoroughly, adjust the pH to 7.4, and bring the volume to 1L.

[0285] Buffer B:

[0286] Sodium phosphate 50mM

[0287] 300mM sodium chloride

[0288] Imidazole 300mM

[0289] Dissolve the substance in approximately 900ml of water, stir thoroughly, adjust the pH to 7.4, and bring the volume to 1L.

[0290] Fixative:

[0291] 50% ethanol

[0292] 7% acetic acid

[0293] Add pure water to a volume of 1L.

[0294] Decolorizing solution:

[0295] 5% ethanol

[0296] Acetic acid 7.5%

[0297] Add pure water to a volume of 1L.

[0298] R-250 stock solution:

[0299]

[0300] 2. Experimental Methods

[0301] 2.1 Cell Culture and Cryopreservation

[0302] 2.1.1 Cell passage

[0303] Remove the cultured cells from the 37℃, 5% CO2 incubator, aspirate the culture medium with a pump, wash 1-2 times with an appropriate amount of PBS solution, add an appropriate amount of trypsin to digest the cells, let them stand in the incubator for 2-5 minutes, observe under a microscope that the cells have become rounded, stop the trypsin digestion with serum-containing culture medium, pipette the adherent cells off the wall, add the cell suspension to a 2ml centrifuge tube, centrifuge at 1000rpm for 3 minutes, then aspirate the supernatant, add fresh culture medium, pipette the cell clusters into a single-cell suspension, and culture in separate trays.

[0304] If the cells are in suspension, add the cell suspension directly to a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, remove the supernatant, wash once with an appropriate amount of PBS, add fresh culture medium, pipette the cell clusters into single-cell suspensions, and culture in separate trays.

[0305] 2.1.2 Cell cryopreservation

[0306] Digest the cells in the culture dish with trypsin, mix thoroughly by pipetting, transfer to centrifuge tubes, and centrifuge at 1000 rpm for 3 min. Discard the supernatant, add an appropriate amount of culture medium containing 10% DMSO, and aliquot into cryovials, 1 ml per tube. Place the cryovials in a 4°C freezer for 30 min, then transfer to a -20°C freezer for 30 min, then transfer to a -80°C freezer for one day, and finally transfer to a liquid nitrogen container for long-term storage.

[0307] 2.1.3 Cell resuscitation

[0308] Remove the cryovials from the liquid nitrogen container and place them in a 37°C water bath to thaw rapidly. Centrifuge at 1000 rpm for 2 minutes. Discard the supernatant, add fresh culture medium preheated to 37°C, mix well by pipetting, and inoculate into petri dishes.

[0309] 2.2 Transformation and Extraction of Recombinant Plasmids

[0310] 2.2.1 Transformation of recombinant plasmids

[0311] (1) Configure the conversion system:

[0312] plasmid 20-30 ng

[0313] 5X KCM 10μl

[0314] Add ddH2O to a volume of 50 μl.

[0315] (2) Add the transformation system to 50 μl of competent cells,

[0316] (3) 42℃ water bath heat shock for 90s.

[0317] (4) Add 800 μl of LB medium (without resistance) and shake at 37°C and 225 rpm for 1 h.

[0318] (5) Centrifuge at 6000 rpm for 5 min, remove 800 μl of supernatant with a pipette, resuspend the precipitate, add it to the corresponding antibiotic plate, and spread the bacterial solution evenly with a spreader. Mix well and place on ice for 20 min.

[0319] (6) Incubate in an inverted incubator at 37℃ for 12-16 hours.

[0320] 2.2.2 Extraction of recombinant plasmids

[0321] The procedure was performed according to the kit instructions.

[0322] 2.3 Cell transfection

[0323] (1) When the cell density reaches about 60%, change the cell medium with serum-free basic medium (DMEM or Opti-MEM) 1 hour before transfection.

[0324] (2) Mix an appropriate amount of the target plasmid DNA to be transfected with an appropriate volume of Opti-MEM medium, then add an appropriate amount of Lipo2000 liposome transfection reagent, mix thoroughly, and let stand at room temperature for 15 minutes. (See Table 2 for the specific amounts of each reagent to be added)

[0325] Table 2. Amounts of reagents used in the liposome method

[0326]

[0327] (3) After standing for 15 minutes, add the mixture to the cell culture dish and place it in a CO2 incubator at 37°C. After 6 hours, replace it with fresh culture medium containing serum. After 12-24 hours, carry out subsequent experiments.

[0328] 2.4 Mammalian One-Hybrid and Reporter Gene Systems

[0329] 2.4.1 Cell Plating

[0330] 293T cells were collected by digestion, at a density of 2-10 × 10⁶ cells per well. 3 One cell line was seeded into a 48-well plate. Before placing the cells into the incubator, gently tap the four edges of the 48-well plate to ensure even distribution of cells in each well, which is beneficial for cell growth. When the cell density reaches approximately 60%, perform the following cell transfection experiment.

[0331] 2.4.2 Transfection

[0332] Prepare the transfection system according to the proportions in Table 2 in 15 ml centrifuge tubes. After standing at room temperature for 15 minutes, add 150 μl of serum-containing culture medium. Aspirate the culture medium from the 48-well plate and add the above mixture to each 48-well plate at 200 μl. Finally, incubate in an incubator for 24 h (5% CO2, 37℃).

[0333] 2.4.3 Dosing

[0334] Approximately 24 hours after transfection, administer the drug according to the pre-designed protocol. First, prepare the drug-containing culture medium, which can be prepared at different concentrations as needed. Then, aspirate the old culture medium from the 48-well plate and add fresh drug-containing medium, 200 μl per well. For each drug and each concentration, generally three replicates are used. A negative control group and a positive control group are required for each experiment.

[0335] 2.4.4 Sample Receiving

[0336] Samples can be collected 12 hours after drug treatment. Observe cell state under an inverted microscope, wash cells twice with 200 μl of pre-cooled PBS, and observe whether the drug affects cell growth. Discard the culture medium and aspirate all PBS. Add 60 μl of 1×Passive Lysis Buffer to each well and lyse cells on a shaker at room temperature for about 30 minutes.

[0337] 2.4.5 Luciferase activity was determined using the Luciferase assay kit.

[0338] Transfer 30 μl of cell lysis supernatant from each well into a 96-well microplate. First, add 50 μl of diluted firefly luciferase substrate and quickly measure the activity of firefly luciferase using a microplate reader. Then, add Renida luciferase substrate and measure its activity. (Because Renida luciferase substrate quenches firefly fluorescence, the two will not interfere with each other. Ensure that the supernatant and luciferase substrate are mixed quickly and thoroughly while maintaining a consistent time interval between adding each sample to the microplate.)

[0339] 2.4.6 Data Analysis

[0340] The ratio of the fluorescence intensity of the firefly luciferase substrate to that of the kidney luciferase substrate is the detection result. The drug activity is compared with that of the negative and positive control groups.

[0341] 2.5 Protein Extraction and Purification in Prokaryotic Systems

[0342] 2.5.1 Optimization of protein induction conditions (taking His tag as an example)

[0343] (1) One colony of each of the control group bacteria (containing the empty pET vector) and the recombinant bacteria (pET vector linked with the target gene) was picked and inoculated into 5 ml of LB medium containing antibiotics and cultured overnight at 37°C and 225 r / m. (Activation of bacterial solution) In addition, the bacterial solution was preserved by adding 600 μl of bacterial solution to 600 μl of 50% sterile glycerol.

[0344] (2) Take 2 ml of the overnight culture and inoculate it into 200 ml of LB medium containing antibiotics. Incubate at 37°C for at least 2 hours until the logarithmic growth phase (OD50). 600 =0.6-0.8).

[0345] (3) Add IPTG to the culture to a final concentration of 1 mM and induce overnight at 16°C (12-16 h).

[0346] Note: The above conditions (final IPTG concentration 1mM, 16℃) are the most commonly used conditions. If induction is not good, you need to explore and optimize the conditions yourself (IPTG 0.01mM-5mM, 16℃-25℃).

[0347] (4) The cultured bacterial solution was placed in a centrifuge tube and centrifuged at 12000g for 5 minutes.

[0348] (5) Add buffer A and 0.1% Triton X-100 (20ml buffer A per L LB bacterial solution) to the bacterial precipitate and resuspend the bacterial precipitate. Under the conditions of 15% power, 3S / 3S, 15min (the ultrasonic conditions need to be determined according to the actual situation, the bacterial solution color turns yellow-brown and changes from viscous to having good fluidity), sonicate in an ice bath to break it up.

[0349] (6) Centrifuge at 18000g, 30min, 4℃ and collect the supernatant.

[0350] (7) Add the supernatant protein from the induced and uninduced steps (6) to the loading buffer for boiling, loading, and gel running (gel concentration depends on the size of the target protein) for identification.

[0351] (8) Coomassie Brilliant Blue R-250 staining, destaining identification (Immerse the gel in fixative, microwave on high for 20 seconds until boiling, remove and discard the fixative. Pour in the staining solution, microwave until boiling under the above conditions, remove and shake for 5 minutes. Discard the staining solution, replace with destaining solution (or tap water) and shake to destain.)

[0352] 2.5.2 Protein Extraction and Expression

[0353] After determining that the induction amount was relatively large, the expression system was scaled up to extract the protein. The method is as follows:

[0354] (1) Take the preserved bacterial culture and inoculate it into 5 ml of LB medium containing antibiotics. Incubate overnight at 37°C and 225 rpm.

[0355] (2) Take 3 ml of overnight culture and inoculate it into 1 L of LB medium containing antibiotics. Incubate at 37°C until the logarithmic growth phase (OD2). 600 =0.6-0.8).

[0356] (3) Induce according to the optimized induction conditions in 2.5.1.

[0357] (4) After induction, transfer the cultured bacterial solution to a centrifuge tube and centrifuge at 12000g for 5 minutes.

[0358] (5) Add buffer A and 0.1% Triton X-100 to the bacterial precipitate to resuspend the bacterial precipitate, and sonicate in an ice bath according to the ultrasonic conditions in 2.5.1.

[0359] (6) Centrifuge at 18000g, 30min, 4℃, and transfer the supernatant into a clean 50ml centrifuge tube.

[0360] (7) Equilibrate His beads. Use a pipette to mix the beads in the reagent bottle. Take 2 ml (2 ml per L of bacterial culture) of the bead suspension into an EP tube. Centrifuge at 3000 rpm and 4℃ for 3 min. Discard the supernatant. Add buffer A and rotate at 4℃ for 10 min. Repeat the above steps 3 times (this step can be performed simultaneously with step (5)).

[0361] (8) Add the balanced beads to the supernatant protein in step (6) and rotate at 4°C for 5 hours.

[0362] (9) Add the mixture to a gasketed chromatography column and collect the beads, ensuring the liquid level remains above the deposited beads. Add washing buffer (composed of 97% buffer A and 3% buffer B) to the column, plug both ends, and rotate at 4°C for 10 minutes. Remove the column, open the plugs, and allow the liquid to flow out naturally. Repeat the above steps four times.

[0363] (10) Add 2-3 ml of buffer B and rotate at 4°C for 1 hour. Collect the liquid and repeat the above steps once. Combine the two collected target protein solutions and run the collected protein solution on a gel, stain it, observe the purity, and use it for subsequent experiments.

[0364] 2.5.3 Coomassie Brilliant Blue Staining

[0365] (1) Soak the protein gel after electrophoresis in fixative, heat it in a microwave oven on high for 20 seconds until it boils, then remove it and discard the fixative.

[0366] (2) Pour in the staining solution, microwave it to boiling under the above conditions, and shake for 5 minutes after removing it.

[0367] (3) Discard the staining solution and replace it with decolorizing solution (or tap water) and shake to decolorize.

[0368] 2.6 Fluorescent Titration

[0369] (1) Adjust the protein concentration to 5 μM (0.25 μg / μl for GST-LBD, 0.125 μg / μl for His-LBD). The protein must be stable at room temperature before titration. Wash the cuvette with ethanol, then with water, and finally rinse with PBS. Add the protein to the cuvette to a total volume of 3 ml and let it stand at room temperature for 10 min.

[0370] (2) Set the excitation wavelength to 280nm and the emission detection wavelength to 300-500nm. The slit wavelengths for excitation and emission are generally set to 5nm and 5nm respectively, but can be adjusted according to specific circumstances. The larger the slit wavelength, the higher the measured peak value, but the accuracy is relatively reduced.

[0371] (3) Begin scanning and record the fluorescence spectrum and peak values. Repeat the measurement three times. If the repeatability of the three measurements is poor, the protein is not yet stable; wait another 5-10 minutes before measuring again. Titration can begin once the repeatability of the three measurements is good (i.e., the peak shapes of the three measurements are basically overlapping).

[0372] (4) Add 3 μl of 1 mM drug to the cuvette (to make the final concentration 1 μM), mix well with a 1 ml pipette, let stand for 5 min, scan, and record the fluorescence spectrum and peak value. Repeat the measurement 3 times to confirm repeatability;

[0373] (5) Repeat step (4) 5 times. Record the fluorescence spectrum and peak values;

[0374] (6) Add 3 μl of 2.5 mM drug to the cuvette (to make a final concentration of 7.5 μM), mix well with a 1 ml pipette, let stand for 5 min, scan, and record the fluorescence spectrum and peak value. Repeat the measurement 3 times to confirm repeatability;

[0375] (7) Repeat the above steps 6 times. Record the fluorescence spectrum and peak values.

[0376] (8)K d Value calculation method

[0377] Calculate K using Origin software according to the following formula. d Value. Using the final concentration of the compound in the solution after each titration as the x-axis and the ΔF after each titration as the y-axis, and fitting the values ​​into the following formula, the K values ​​for proteins and small molecules can be obtained. d value.

[0378] y = C ＊ (x+P+K d ) / 2-C ＊ sqrt((x+P+K d )^2-4 ＊ P ＊ x) / 2

[0379] In the formula, y is the fluorescence change or fluorescence quenching value (ΔF) after each titration, x is the final concentration of the compound in the solution after each titration, C is the theoretical maximum quenching fluorescence value / total protein, and P is the protein concentration used (μM, 5 in this experiment).

[0380] 2.7 Extraction and Western Blot of Mouse Hippocampal Proteins

[0381] (1) Sample collection: The hippocampus was isolated from the mouse brain;

[0382] (2) Tissue lysis: After the hippocampus was broken up, it was lysed for 30 min with 30-100 μl of RIPA cell lysis buffer (depending on the number of cells). During this time, the mixture was shaken for 10 s every 5 minutes on a shaker. Then it was centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was collected and the protein concentration was measured.

[0383] RIPA lysis buffer formulation: 50mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150mM NaCl (add 1mM EDTA, 1mM MPMSF, 1mM NaF, 1mM NaVO3 and a mixture of protease inhibitors (cocktail) just before use).

[0384] (3) Determination of protein concentration

[0385] Protein electrophoresis: Take 20-100 μg of protein sample (depending on protein abundance in cells), add an equal volume of 2× Loading buffer (containing 10% β-mercaptoethanol), boil at 100℃ for 10 min, centrifuge briefly, and then perform electrophoresis in Tris-glycine buffer (80V, switch to 120V after passing through the separating gel). Add 5 μl of protein marker to each electrophoresis plate. Electrophoresis buffer formulation: Weigh 3.03 g of Tris-HCl, 18.77 g of Glycine, and 1 g of SDS and dissolve in 1000 ml of distilled water.

[0386] Electrotransfer: The electrotransfer solution was pre-cooled at 4℃. After cutting the gel, PVDF membranes of the same size (pre-soaked in anhydrous methanol for 1 min) and filter paper were pre-soaked in the electrotransfer solution. After attaching the PVDF membrane to the gel, air bubbles were removed. Filter paper was then placed on both sides, and air bubbles were removed. The membranes were placed in the electrotransfer tank with the positive electrode facing outwards. Electrotransfer solution and ice packs were added, and electrotransfer was performed at 4℃ (100V, 60 min or 300mA, 90 min). Electrotransfer solution formulation: Weigh 11.25g of Glycine and 2.425g of Tris-HCl, dissolve them in 900ml of distilled water, and then add 100ml of methanol.

[0387] Antigen-antibody reaction:

[0388] ①The PVDF membrane after electroporation is sealed and shaken in 5% skim milk sealing solution for 1 hour or longer;

[0389] ② Primary antibody reaction: After blocking, the membrane is sealed in a hybridization bag, and the reaction is carried out at 0.1 ml / cm². 2Add primary antibody (concentration from 1:500 to 1:2000, depending on the antibody) and incubate overnight on a shaker at 4°C or shake for 3 hours at room temperature;

[0390] ③ Secondary antibody reaction: Wash the membrane three times in TBST, 5 min each time, and then shake for 1 hour at room temperature in an appropriate secondary antibody solution (the secondary antibody is dissolved in the blocking buffer at a concentration of 1:8000 to 1:20000). TBST formulation: Weigh 1.21 g of Tris-HCl and 8.77 g of NaCl, dissolve them in 1000 ml of distilled water, adjust the pH to 7.6, and then add 1 ml of Tween-20.

[0391] (7) ECL detection: Discard the secondary antibody, wash the membrane with TBST, shake three times at room temperature for 5 minutes each time, with the protein side facing up, place it in plastic wrap, mix ECL (Pierce) solution A and solution B 1:1 (V / V), and in the dark, drop it onto the membrane surface at a rate of 0.05-0.1 ml / cm2, incubate for 1 minute, absorb excess liquid with filter paper, wrap the membrane with plastic wrap, and immediately expose it in the dark for several seconds or longer (depending on the fluorescence intensity).

[0392] 2.9 AD mouse model tissue section sample detection

[0393] 2.9.1 Experimental Model:

[0394] APP / PS1 transgenic mice aged 7-9 months were randomly selected and administered Z-10, Z-36 (40 mg / kg / day) or blank control solution orally for 15 consecutive days. Hippocampal tissue of the mice was collected.

[0395] 2.9.2 Preparation of Frozen Tissue Sections

[0396] 1. Mouse cardiac perfusion: 12-month-old APP / PS1 double-positive mice and control C57-negative mice were anesthetized by injection of 10% chloral hydrate (400 mg / kg); the left ventricle was punctured and perfused with physiological saline (3‰ heparin), while the right atrial appendage was cut open to drain blood until the outflowing fluid was colorless, at which point perfusion was stopped. Brain tissue was removed, trimmed with a scalpel, and the olfactory bulb and cerebellum were removed.

[0397] 2. Quick-freezing brain tissue: The extracted brain tissue is embedded in gel, and a small square box is folded from aluminum foil. The trimmed brain tissue is placed inside, and an appropriate amount of OCT gel is added to completely immerse the tissue. The tissue is then transferred to a plastic box, and the box is held firmly with tweezers and slowly placed horizontally into a beaker containing liquid nitrogen. When the bottom of the box touches the liquid nitrogen, vaporization and boiling begin. At this point, the small box should remain in place and not be immersed in the liquid nitrogen. The tissue will rapidly freeze into a block in approximately 30 seconds. After freezing, the tissue can be sectioned in a cryogenic chamber or stored at -80°C.

[0398] 3. Sectioning: Coat a layer of OCT embedding gel on the sample holder, place the tissue block on it, and put it in a cryostat (-20℃) for 20 to 30 minutes; adjust the position and angle of the blade and anti-roll plate, and slice to a thickness of 8μm; attach the tissue section to a glass slide, mark it, and immediately fix it in acetone at 4℃. After 15 minutes, take it out and dry it at room temperature for 2 hours, and store it at -80℃.

[0399] 2.10.4 Immunostaining of frozen tissue sections

[0400] 1. Remove the sections from the -80℃ freezer, allow them to warm to room temperature for half an hour, then fix them in acetone at 4℃ for 20 minutes. Wash gently with PBS three times, 5 minutes each time.

[0401] 2. Circle the tissue to be stained with a histochemical pen, add 1% Triton X-100 (prepared with PBS), allow to permeate at room temperature for 45 min, and wash with PBS 3 times, 5 min each time;

[0402] 3. Add 1% BSA (prepared with PBS) and block at 37°C for 30 min;

[0403] 4. Primary antibody incubation: Dilute the primary antibody with PBS to a dilution of 1:200, incubate overnight at 4°C, and wash three times with PBS for 5 minutes each time;

[0404] 5. Add fluorescent secondary antibody at a dilution of 1:200, incubate at 37°C in the dark for 2 hours, and wash three times with PBS for 5 minutes each time;

[0405] 6. Add DAPI solution, 2 μg / mL, incubate at 37°C in the dark for 5 min, wash 3 times with PBS, 5 min each time;

[0406] 7. Sealed with neutral resin;

[0407] 8. Imaging with a fluorescence microscope.

[0408] II. Biological Experiments

[0409] 1. Determination of the binding of Z-10 series compounds to RXRα protein by fluorescence titration.

[0410] (1) The protein was incubated with 0, 1, 2, 3, 4, 5, 7.5, 10, 12.5 and 15 μM Z-10 and purified 5 μM RXRα / LBD protein, respectively. The emission spectra of RXRα / LBD protein excited by 280 nm excitation light in the range of 300-450 nm were detected, and the results were analyzed using Origin software and the formula "y = C ＊ (x+5+K d ) / 2-C ＊ sqrt(x+5+K d ) 2 -4 ＊ 5＊ The Kx of the combination of compound Z-10 and RXRα was calculated using x) / 2”. d Value, result as Figure 3 As shown in A and 3B.

[0411] The results showed that Z-10 binds to RXRα, and the K-type of Z-10 and RXRα binds to it. d The value is 0.69±0.15μM.

[0412] (2) Referring to the above method, calculate the Kα of compounds Z-21, Z-37, Z-22, Z-23, Z-46 and Z-47 when they bind to RXRα. d Value, result as Figure 3 As shown in C.

[0413] The results showed that the Z-10 derivatives Z-37 and Z-23, modified with methyl and methoxy groups at the 2-position, and the Z-10 derivatives Z-21 and Z-22, modified with methyl and methoxy groups at the 4-position, all bound RXRα. The binding K values ​​of each compound were... d The values ​​were: Z-37: 4.89±0.54μM; Z-23: 0.68±0.28μM; Z-21: 12.41±1.74μM; Z-22: 18.23±4.65μM.

[0414] The Z-10 derivatives Z-46 and Z-47, which are modified with hydroxyl groups at positions 2 and 4, respectively, showed no binding to RXRα.

[0415] Therefore, methyl and methoxy modifications at the 2-position are more conducive to the binding of the compound with RXRα than methyl and methoxy modifications at the 4-position. Hydroxyl modifications at the 2- and 4-positions are both detrimental to the binding of the compound with RXRα.

[0416] (3) The protein was incubated with Z-36 at concentrations of 0, 1, 2, 3, 4, 5, 7.5, 10, 12.5, 15, 17.5, and 20 μM, respectively, and purified RXRα / LBD protein at 5 μM. The emission spectra of RXRα / LBD protein excited by 280 nm excitation light in the 300-450 nm range were detected, and the results were analyzed using Origin software and the formula "y = C ＊ (x+5+K d ) / 2-C ＊ sqrt(x+5+K d ) 2 -4 ＊ 5 ＊ The Kx of the combination of compound Z-36 and RXRα was calculated using x) / 2”. d Value, result as Figure 5 As shown in E and 5F.

[0417] The results showed that Z-36 combined with RXRα, and the K of Z-36 and RXRα combined d The value is 0.38±0.17μM.

[0418] 2. Effects of Z-10 series compounds on the transcriptional activity of RXRα and its dimers determined by yeast one-hybrid assay and reporter gene assay.

[0419] (1) HEK 293T cells were transfected with pG5-luciferase and pBIND-RXRαLBD plasmids for 24 hours, followed by treatment with DMSO (as negative control), 5 μM Z-10, Z-21, Z-22, Z-23, Z-37, Z-46 or Z-47, or 0.1 μM 9-cis-RA (9-c-RA) (as positive control) for 12 hours. Reporter gene activity was measured using firefly and Renidae dual-luciferase systems. Results are shown in […]. Figure 2 A.

[0420] The results showed that Z-10, Z-37, and Z-23 significantly activated the transcriptional activity of Gal4 / DBD-RXRα / LBD, while Z-21, Z-22, Z-46, and Z-47 did not significantly activate the transcriptional activity of Gal4 / DBD-RXRα / LBD under these conditions.

[0421] Therefore, methyl and methoxy modifications at the 2-position are more conducive to the activation of transcriptional activity of Gal4 / DBD-RXRα / LBD than methyl and methoxy modifications at the 4-position. Hydroxyl modifications at both the 2- and 4-positions are detrimental to the activation of transcriptional activity of Gal4 / DBD-RXRα / LBD.

[0422] (2) HEK 293T cells were transfected with pG5-luciferase and pBIND-RARαLBD plasmids for 24 hours, followed by treatment with DMSO (as a negative control), 5 μM Z-10, Z-21, Z-22, Z-23, Z-37, Z-46 or Z-47, or 0.1 μM ATRA (as a positive control) for 12 hours. Reporter gene activity was measured using firefly and Renidae dual-luciferase systems. Results are shown in […]. Figure 2 B.

[0423] The results showed that Z-10, Z-21, Z-22, Z-23, Z-37, Z-46 and Z-47 could not significantly activate the transcriptional activity of Gal4 / DBD-RARα / LBD.

[0424] (3) HEK 293T cells were transfected with pG5-luciferase and pBIND-ERαLBD plasmids for 24 hours, followed by treatment with DMSO (as a negative control), 5 μM Z-10, Z-21, Z-22, Z-23 or Z-37, or 10 nM Estradiol (E2) (as a positive control) for 12 hours. Reporter gene activity was measured using firefly and Renidae dual-luciferase systems. Results are shown in […]. Figure 2 C.

[0425] The results showed that Z-10, Z-21, Z-22, Z-23, Z-37, Z-46 and Z-47 could not significantly activate the transcriptional activity of Gal4 / DBD-ERα / LBD.

[0426] (4) Reporter gene therapy was used to co-transfect HEK293T cells with pGL6-TA-RXRE-luciferase, renilla-luciferase, and pCMV-myc-RXRα plasmids for 24 hours. Subsequently, the cells were treated for 12 hours with DMSO (as a negative control), 5 μM Z-10, Z-21, Z-22, Z-23, Z-37, Z-46, or Z-47, or 0.1 μM 9-cis-RA (9-c-RA) (as a positive control). Reporter gene activity was measured using a firefly and Renilla dual-luciferase system. The results are shown in […]. Figure 2 D.

[0427] The results showed that Z-10, Z-21, Z-22, Z-46 and Z-47 did not significantly activate the transcriptional activity of RXRα / RXRα homodimer, while Z-37 and Z-23 significantly activated the transcriptional activity of RXRα / RXRα homodimer.

[0428] Therefore, methyl and methoxy modifications at the 2-position are more conducive to activating the transcriptional activity of the RXRα / RXRα homodimer than methyl and methoxy modifications at the 4-position. Both hydroxyl modifications at the 2- and 4-positions are detrimental to activating the transcriptional activity of the RXRα / RXRα homodimer.

[0429] (5) Using reporter gene technology, HEK293T cells were co-transfected with pGL6-TA-RARE-luciferase, renilla-luciferase, pCMV-myc-RXRα, and pCMV-myc-RARα plasmids for 24 hours. Subsequently, the cells were treated with DMSO, 5 μM Z-10, Z-21, Z-22, Z-23, Z-37, Z-46, or Z-47, and / or 0.1 μM ATRA for 12 hours. Reporter gene activity was measured using a firefly and Renidae dual-luciferase system. The results are shown in […]. Figure 2 E.

[0430] The results showed that Z-10, Z-21, Z-22, Z-23, Z-37, Z-46, and Z-47 alone could not significantly activate the transcriptional activity of the RXRα / RARα heterodimer. Z-23 and Z-37 significantly enhanced the ATRA-activated transcriptional activity of the RXRα / RARα heterodimer, while Z-10, Z-21, Z-22, Z-46, and Z-47 could not significantly enhance the ATRA-activated transcriptional activity of the RXRα / RARα heterodimer. The DMSO treatment group (first column) and the ATRA treatment group (second column) served as control groups.

[0431] Therefore, methyl and methoxy modifications at the 2-position are more beneficial than those at the 4-position in enhancing the transcriptional activity of ATRA in activating the RXRα / RARα heterodimer. Both hydroxyl modifications at the 2- and 4-positions are detrimental to enhancing the transcriptional activity of ATRA in activating the RXRα / RARα heterodimer.

[0432] (6) Using reporter gene technology, Cos-7 cells were co-transfected with pGL6-TA-PPRE-luciferase, renilla-luciferase, pCMV-myc-RXRα, and pCMV-myc-PPARγ plasmids for 24 hours. Subsequently, the cells were treated with DMSO, 5 μM Z-10, Z-21, Z-22, Z-23, Z-37, Z-46, or Z-47, and / or 0.5 μM rosiglitazone (Rosi) for 12 hours. Reporter gene activity was measured using a firefly and kidney-tonifying dual-luciferase system. The results are shown in […]. Figure 2 F.

[0433] The results showed that Z-10, Z-23, and Z-37 alone significantly activated the transcriptional activity of the RXRα / PPARγ heterodimer, while Z-21, Z-22, Z-46, and Z-47 alone did not significantly activate the transcriptional activity of the RXRα / PPARγ heterodimer. Z-10, Z-23, and Z-37 significantly enhanced the rosiglitazone-activated transcriptional activity of the RXRα / PPARγ heterodimer, while Z-21, Z-22, Z-46, and Z-47 did not significantly enhance the rosiglitazone-activated transcriptional activity of the RXRα / PPARγ heterodimer. The DMSO treatment group (first column) and the rosiglitazone (Rosi) treatment group (second column) served as control groups.

[0434] Therefore, methyl and methoxy modifications at the 2-position are more conducive to the activation of the transcriptional activity of the RXRα / PPARγ heterodimer than methyl and methoxy modifications at the 4-position, and also enhance the activation of the RXRα / PPARγ heterodimer by rosiglitazone. Hydroxyl modifications at the 2- and 4-positions are detrimental to the activation of the RXRα / PPARγ heterodimer, and also detrimental to enhancing the activation of the RXRα / PPARγ heterodimer by rosiglitazone.

[0435] (7) Using reporter gene technology, HEK 293T cells were co-transfected with pGL6-TA-LXRE-luciferase, renilla-luciferase, pCMV-myc-RXRα, and pCMV-myc-LXRα plasmids for 24 hours. Subsequently, the cells were treated with DMSO, 5 μM Z-10, Z-21, Z-22, Z-23, Z-37, Z-46, or Z-47, and / or 0.25 μM T0901317 (T090) for 12 hours. Reporter gene activity was measured using a firefly and Renidae dual-luciferase system. The results are shown in […]. Figure 2 G.

[0436] The results showed that Z-10, Z-23, and Z-37 alone significantly activated the transcriptional activity of the RXRα / LXRα heterodimer, while Z-21, Z-22, Z-46, and Z-47 alone did not significantly activate the transcriptional activity of the RXRα / LXRα heterodimer. Z-10, Z-23, and Z-37 significantly enhanced the transcriptional activity of T0901317 in activating the RXRα / LXRα heterodimer, while Z-21, Z-22, Z-46, and Z-47 did not significantly enhance the transcriptional activity of T0901317 in activating the RXRα / LXRα heterodimer. The DMSO treatment group (first column) and the T0901317 (T090) treatment group (second column) served as control groups.

[0437] Therefore, methyl and methoxy modifications at the 2-position are more conducive to the activation of the transcriptional activity of the RXRα / LXRα heterodimer than methyl and methoxy modifications at the 4-position, and also enhance the activation of the RXRα / LXRα heterodimer by T0901317. Hydroxyl modifications at the 2- and 4-positions are detrimental to the activation of the RXRα / LXRα heterodimer, and also detrimental to enhancing the activation of the RXRα / LXRα heterodimer by T0901317.

[0438] 3. Effects of compound concentration on the transcriptional activity of RXRα and its dimer

[0439] (1) Using yeast one-hybrid assay, HEK 293T cells were transfected with pG5-luciferase and pBIND-RXRαLBD plasmids for 24 hours. Subsequently, the cells were treated for 12 hours with DMSO (as a negative control), 5, 10, or 15 μM of Z-10, Z-21, or Z-22, or 9-cis-RA (9-c-RA, 0.1 μM) (as a positive control). Reporter gene activity was measured using firefly and Renalis dual-luciferase systems. The results are shown in […]. Figure 3 D.

[0440] The results showed that Z-10, Z-21, or Z-22 could activate the transcriptional activity of Gal4 / DBD-RXRα / LBD in a concentration-dependent manner. However, at the same concentration, the activation ability of Z-21 and Z-22 modified with methyl and methoxy groups at the 4-position was weaker than that of Z-10. Therefore, modification with methyl and methoxy groups at the 4-position is detrimental to the activation of the transcriptional activity of Gal4 / DBD-RXRα / LBD by the compounds.

[0441] (2) Using reporter gene technology, HEK 293T cells were co-transfected with pGL6-TA-RXRE-luciferase, renilla-luciferase, and pCMV-myc-RXRα plasmids for 24 hours. Subsequently, the cells were treated with Z-10, Z-23, or Z-36 at 0.5, 1, 2, 3, 4, 5, or 7.5 μM for 12 hours. Reporter gene activity was measured using firefly and Renilla dual-luciferase systems. Results are shown in […]. Figure 4 D.

[0442] The results showed that Z-10 could not activate the transcriptional activity of the RXRα / RXRα homodimer, while Z-36 and Z-23 could activate the transcriptional activity of the RXRα / RXRα homodimer in a concentration-dependent manner. Therefore, modification at the 2-position with methoxy and ethoxy groups is beneficial for the compounds to activate the transcriptional activity of the RXRα / RXRα homodimer.

[0443] (3) Using reporter gene therapy, HEK 293T cells were transfected with pGL6-TA-LXRE-luciferase, renilla-luciferase, pCMV-myc-RXRα, and pCMV-myc-LXRα plasmids for 24 hours. The cells were then treated with 0.5, 1, 2, 3, 4, or 5 μM Z-10, Z-23, or Z-36 for 12 hours, respectively. Reporter gene activity was measured using a firefly and Renidae dual-luciferase system. Results are shown in […]. Figure 5 A.

[0444] The results showed that Z-10, Z-23, and Z-36 all activated the transcriptional activity of the RXRα / LXRα heterodimer in a concentration-dependent manner, with Z-23 and Z-36 exhibiting significantly stronger activation effects than Z-10. Therefore, modification at the 2-position with methoxy and ethoxy groups is beneficial for the compounds to activate the transcriptional activity of the RXRα / LXRα heterodimer.

[0445] (4) Using reporter gene technology, Cos-7 cells were transfected with pGL6-TA-PPRE-luciferase, renilla-luciferase, pCMV-myc-RXRα and pCMV-myc-PPARγ plasmids for 24 hours, and then treated with 0.5, 1, 2, 3, 4 or 5 μM Z-10, Z-23 or Z-36 for 12 hours. The reporter gene activity was measured by firefly and Renal luciferase dual-luciferase system. The results are shown in 5B.

[0446] The results showed that Z-10, Z-23, and Z-36 all activated the transcriptional activity of the RXRα / PPARγ heterodimer in a concentration-dependent manner, with Z-23 and Z-36 showing significantly stronger activation than Z-10. Therefore, modification at the 2-position with methoxy and ethoxy groups is beneficial for the compounds to activate the transcriptional activity of the RXRα / PPARγ heterodimer.

[0447] 4. Effects of alkoxy modification at the 2-position of compound Z-10 on the transcriptional activity of RXRα and its dimer

[0448] (1) Yeast one-hybrid assay was used to detect the activation of RXRα transcriptional activity by Z-10 series compounds modified at the 2-alkoxy position. HEK 293T cells were transfected with pG5-luciferase and pBIND-RXRαLBD plasmids for 24 hours, and then treated for 12 hours with DMSO (as a negative control), 5 μM of Z-10, Z-23, Z-36, Z-49, Z-52, or Z-53, or 0.1 μM of 9-cis-RA (as a positive control). Reporter gene activity was measured using firefly and Renalis dual-luciferase systems. The results are shown in […]. Figure 4 A.

[0449] The results showed that Z-10, Z-23, Z-36, Z-49, Z-52, and Z-53 all significantly activated Gal4 / DBD-RXRα / LBD transcriptional activity. Therefore, alkoxy modification at the 2-position is beneficial for the compounds to activate Gal4 / DBD-RXRα / LBD transcriptional activity.

[0450] (2) Yeast one-hybrid assay was used to detect the activation of RARα transcriptional activity by Z-10 series compounds modified at the 2-alkoxy position. HEK 293T cells were transfected with pG5-luciferase and pBIND-RARαLBD plasmids for 24 hours, and then treated for 12 hours with DMSO (as a negative control), 5 μM of Z-10, Z-23, Z-36, Z-49, Z-52, or Z-53, or 0.1 μM ATRA (as a positive control). Reporter gene activity was measured using firefly and Renalis dual-luciferase systems. The results are shown in […]. Figure 4 B.

[0451] The results showed that Z-10, Z-23, Z-36, Z-49, Z-52, and Z-53 could not significantly activate Gal4 / DBD-RARα / LBD transcriptional activity. Therefore, compounds modified with an alkoxy group at the 2-position cannot activate Gal4 / DBD-RARα / LBD transcriptional activity.

[0452] (3) Reporter gene assay was used to detect the activation of RXRα homodimer transcriptional activity by Z-10 series compounds modified at the 2-alkoxy position. HEK 293T cells were transfected with pGL6-TA-RXRE-luciferase, renilla-luciferase, and pCMV-myc-RXRα plasmids for 24 hours. After treatment with DMSO (as a negative control), 5 μM of Z-10, Z-23, Z-36, Z-49, Z-52, or Z-53, or 0.1 μM of 9-cis-RA (as a positive control) for 12 hours, the reporter gene activity was measured using firefly and Renalis dual-luciferase systems. The results are shown in […]. Figure 4 C.

[0453] The results showed that Z-10 could not significantly activate the transcriptional activity of the RXRα / RXRα homodimer, while Z-23, Z-36, Z-49, Z-52, and Z-53 could all significantly activate the transcriptional activity of the RXRα / RXRα homodimer. Therefore, alkoxy modification at the 2-position is beneficial for the compounds to activate the transcriptional activity of the RXRα / RXRα homodimer.

[0454] 5. Effects of Z-10 series compounds combined with nuclear receptor ligands on the transcriptional activity of RXRα heterodimers

[0455] This experiment used reporter gene therapy to investigate the effect of combining a nuclear receptor ligand that forms a heterodimer with RXRα on the transcriptional activity of the dimer.

[0456] (1) HEK293T cells were transfected with pGL6-TA-RARE-luciferase, renilla-luciferase, pCMV-myc-RXRα, and pCMV-myc-RARα plasmids for 24 hours. After treatment with DMSO, 5 μM Z-10, Z-23, Z-36, Z-49, Z-52, or Z-53, or / and 0.1 μM ATRA for 12 hours, the reporter gene activity was measured using a firefly and Renidae dual-luciferase system. Results are shown in […]. Figure 4 E.

[0457] The results showed that Z-10, Z-23, Z-36, Z-49, Z-52, and Z-53 alone could not significantly activate the transcriptional activity of the RXRα / RARα heterodimer. Z-23, Z-36, and Z-49 significantly enhanced the ATRA-activated transcriptional activity of the RXRα / RARα heterodimer, while Z-10, Z-52, and Z-53 could not significantly enhance the ATRA-activated transcriptional activity of the RXRα / RARα heterodimer. The DMSO-treated group (first column) and the ATRA-treated group (second column) served as control groups. Therefore, modification at the 2-methoxy, ethoxy, and isopropoxy positions is beneficial for compounds to enhance the ATRA-activated transcriptional activity of the RXRα / RARα heterodimer.

[0458] (2) HEK 293T cells were transfected with pGL6-TA-LXRE-luciferase, renilla-luciferase, pCMV-myc-RXRα, and pCMV-myc-LXRα plasmids for 24 hours. After treatment with DMSO, 5 μM of Z-series compounds Z-10, Z-23, Z-36, Z-49, Z-52, or Z-53, and 0.25 μM T0901317 (T090) for 12 hours, the reporter gene activity was measured using a firefly and Renidae dual-luciferase system. Results are shown in […]. Figure 5 C.

[0459] The results showed that Z-10 did not significantly enhance the transcriptional activity of T0901317 in activating the RXRα / LXRα heterodimer, while Z-23, Z-36, Z-49, Z-52, and Z-53 all significantly enhanced this activity. Furthermore, Z-36 was more potent than Z-23, and Z-23 was more potent than Z-10. The DMSO treatment group (first column) and the T0901317 (T090) treatment group (second column) served as control groups. Therefore, modification at the 2-position with methoxy, ethoxy, isopropoxy, propoxy, or isobutoxy compounds is beneficial in enhancing the transcriptional activity of T0901317 in activating the RXRα / LXRα heterodimer.

[0460] (3) Cos-7 cells were transfected with pGL6-TA-PPRE-luciferase, renilla-luciferase, pCMV-myc-RXRα, and pCMV-myc-PPARγ plasmids for 24 hours. After treatment with DMSO, 5 μM Z-series compounds Z-10, Z-23, Z-36, Z-49, Z-52, or Z-53, and 0.5 μM rosiglitazone (Rosi) for 12 hours, respectively, the reporter gene activity was measured using a firefly and kidney dual-luciferase system. The results are shown in […]. Figure 5 D.

[0461] The results showed that Z-52 and Z-53 did not significantly enhance the transcriptional activity of rosiglitazone in activating the RXRα / PPARγ heterodimer, while Z-10, Z-23, Z-36, and Z-49 all significantly enhanced this activity, with Z-36 showing a stronger effect than Z-23, and Z-23 showing a stronger effect than Z-10. The DMSO treatment group (first column) and the rosiglitazone (Rosi) treatment group (second column) served as control groups. Therefore, modification at the 2-ethoxy and methoxy positions is beneficial for compounds to enhance the transcriptional activity of rosiglitazone in activating the RXRα / PPARγ heterodimer.

[0462] The effects of Z-10, Z-21, Z-22, Z-23, Z-37, Z-46, or Z-47, in combination with ATRA, Rosi, or T0901317, on the transcriptional activity of RXRα / RARα, RXRα / PPARγ, or RXRα / LXRα dimers can be seen in sections 2(5)-2(7) above. Figure 2 E, 2F, 2G.

[0463] 6. Effects of Z-10 series compounds on Aβ plaques in the hippocampus of AD model mice

[0464] APP / PS1 transgenic female and male mice were administered corn oil (Vehicle), Z-10, and Z-36 by gavage for 15 days at 40 mg / kg / day, respectively. Hippocampal samples were then removed for Aβ immunofluorescence staining, and the number of Aβ plaques was counted. Results are shown below. Figure 6 and Figure 7 .

[0465] The results showed that Z-10 and its 2-ethoxy modified derivative Z-36 could reduce Aβ plaques in the hippocampus of APP / PS1 transgenic AD model mice, and the effect of Z-36 was stronger than that of Z-10.

[0466] 7. Effects of Z-10 series compounds on Aβ plaques in the cerebral cortex of AD model mice

[0467] APP / PS1 transgenic female and male mice were administered corn oil (Vehicle), Z-10, and Z-36 by gavage at 40 mg / kg / day for 15 days. Cerebral cortex samples were extracted for Aβ immunofluorescence staining, and the number of Aβ plaques was counted. Results are shown below. Figure 8 and Figure 9 .

[0468] The results showed that Z-10 and its 2-ethoxy modified derivative Z-36 could reduce Aβ plaques in the cerebral cortex of APP / PS1 transgenic AD model mice, and the effect of Z-36 was stronger than that of Z-10.

[0469] 8. Induction of ABCA1 and ABCG1 proteins in the hippocampus of AD model mice by Z-10 series compounds

[0470] APP / PS1 transgenic mice were administered corn oil (Vehicle), Z-10, and Z-36 by gavage for 15 days at 40 mg / kg / day. Hippocampal tissue was then extracted for Western blotting to detect the expression of ABCA1, ABCG1, and apoE. Gray-scale analysis of the immunoblot bands was performed. Results are shown below. Figure 10 .

[0471] The results showed that Z-10 and its 2-ethoxy modified derivative Z-36 induced the expression of ABCA1 and ABCG1 in the hippocampus of APP / PS1 transgenic AD mice, and the effect of Z-36 was stronger than that of Z-10.

[0472] III. Preparation of Compounds

[0473] The compounds in this application can be prepared by the following methods.

[0474] Synthetic method:

[0475]

[0476] The definitions of each group are as described above.

[0477] Weigh 5 mmol of aromatic aldehyde II-1 and 0.5 mmol of ammonium acetate into a reaction flask / thick-walled pressure-resistant tube, then add 5 mL of nitromethane and 5 mL of toluene sequentially and mix thoroughly. Heat under reflux at 70-80 °C for 12 h in a constant-temperature oil bath. Monitor the reaction using thin-layer chromatography to see if the starting material spot disappears completely. If it does not disappear completely, continue the reaction until it disappears completely or stops decreasing. After the reaction is complete, add 20 mL of saturated NaCl solution and extract three times with 30 mL of ethyl acetate. Combine the organic phases, wash with 30 mL of saturated brine, dry with anhydrous sodium sulfate, and remove the solvent by rotary evaporation under reduced pressure. Mix the residue with 100-200 mesh silica gel and separate by medium-pressure preparation or silica gel column chromatography to obtain the pure product for compound structure identification.

[0478] When R1 (or R3) is C 1-4 When alkoxylated, intermediate II-1 can be prepared from II-2 via an O-alkylation reaction; wherein R1' is a hydroxyl group (or hydrogen) and R3' is hydrogen (or hydroxyl group):

[0479]

[0480] O-alkylation reactions can be carried out using methods commonly used in the art, for example: in the presence of a strong base (e.g., NaH, K₂CO₃, potassium alkoxide, sodium alkoxide, etc., 1-2.5 eq.), the compound of formula II-2 reacts with an alkylating agent (e.g., C 1-4 Compound II-1 was prepared by reacting haloalkyl and sulfate diester compounds (1-2.5 eq.) at room temperature or under heating conditions (40-80 °C) and then post-treatment.

[0481] 1. (E)-4-(2-nitrovinyl)naphthyl-1-phenol (Z46)

[0482]

[0483] Prepared according to the general synthetic method, with a separation yield of 79%. Yellow solid.

[0484] 1 H NMR(600MHz,CHLOROFORM-d)δppm 6.48(br.s.,1H)6.88(d,J=8.07Hz,1H)7.59(t,J=1.00Hz,1H)7.64(d,J=13.39Hz,1H)7.66-7.69( m,1H)7.70(d,J=8.07Hz,1H)8.13(d,J=8.44Hz,1H)8.31(d,J=8.25Hz,1H)8.81(d,J=13.39Hz,1H).

[0485] 13 C NMR(151MHz,CHLOROFORM-d)δppm 108.75(s,1C)119.34(s,1C)122.80(s,1C)122.91(s,1C)124.61(s,1C)126.09(s,1C) 127.89(s,1C)128.51(s,1C)133.25(s,1C)136.18(s,1C)136.26(s,1C)156.04(s,1C).

[0486] HRMS(ESI)calcd for C 12 H8NO3 - [MH] - :214.0510,found:214.0501.

[0487] 2. (E)-2-Isopropoxy-1-(2-nitrovinyl)naphthalene (Z49)

[0488]

[0489] (1) Synthesis of (E)-2-isopropoxy-1-(2-nitrovinyl)naphthalene

[0490] The raw material 2-hydroxy-1-naphthaldehyde and iodoethane were added to DMF solution and reacted overnight at 70°C under the catalysis of potassium carbonate to synthesize 2-isopropoxy-1-naphthaldehyde.

[0491] (2) Synthesis of Z49.

[0492] Prepared according to the general synthetic method, with a separation yield of 59%. Yellow solid.

[0493] 1 H NMR(600MHz,CHLOROFORM-d)(ppm)1.49(d,J=6.05Hz,6H)4.90(spt,J=6.08Hz,1H)7.29(d,J=9.17Hz,1H)7.41-7.46(m,1H)7.60(ddd,J=8. 48,7.01,1.19Hz,1H)7.81(d,J=8.07Hz,1H)7.93(d,J=9.17Hz,1H)8.16(d,J=8.62Hz,1H)8.20(d,J=13.20Hz,1H)8.84(d,J=13.39Hz,1H).

[0494] 13C NMR(151MHz,CHLOROFORM-d)(ppm)22.41(s,2C)72.18(s,1C)112.41(s,1C)114.27(s,1C)122.18(s,1C)124.44(s,1C )128.36(s,1C)128.75(s,1C)128.96(s,1C)131.22(s,1C)133.62(s,1C)134.14(s,1C)140.03(s,1C)157.63(s,1C).

[0495] HRMS(ESI)calcd for C 15 H 16 NO3 + [M+H] + :258.1125,found:258.1123.

[0496] 3.(E)-2-propoxy-1-(2-nitrovinyl)naphthalene

[0497]

[0498] Prepared according to the Z-49 synthesis method, separation yield: 51%. Orange solid.

[0499] 1 H NMR(600MHz,CHLOROFORM-d)(ppm)1.13(t,J=7.43Hz,3H)1.98(sxt,J=7.08Hz,2H)4.23(t,J=6.51Hz,2H)7.29(d,J=8.99Hz,1H)7.43(t,J=7 .52Hz,1H)7.60(ddd,J=8.53,7.06,1.28Hz,1H)7.94(d,J=9.17Hz,1H)8.16(d,J=8.62Hz,1H)8.18(d,J=13.20Hz,1H)8.84(d,J=13.20Hz,1H)

[0500] 13 C NMR(151MHz,CHLOROFORM-d)ppm 10.75(s,1C)22.72(s,1C)71.21(s,1C)111.56(s,1C)113.14(s,1C)122.19(s,1C)124.42(s,1C)128.46( s,1C)128.84(s,1C)129.05(s,1C)131.04(s,1C)133.48(s,1C)134.38(s,1C)140.04(s,1C)158.61(s,1C)

[0501] HRMS(ESI)calcd for C15H16NO3+[M+H]+:258.1125,found:258.1124.

[0502] 4. (E)-1-(nitrovinyl)-2-(2-butoxy)naphthalene (Z-53)

[0503]

[0504] Prepared according to the Z-49 synthesis method, with a separation yield of 45%, it is a yellow oily liquid.

[0505] 1 H NMR(600MHz,CHLOROFORM-d)(ppm)1.04(t,J=7.52Hz,3H)1.43(d,J=6.05Hz,3H)1.75- 1.83(m,1H)1.87-1.96(m,1H)4.65-4.72(m,1H)7.29(d,J=9.17Hz,1H)7.43(ddd,J=7.9 8,6.97,0.83Hz,1H)7.60(ddd,J=8.53,6.97,1.38Hz,1H)7.81(d,J=8.07Hz,1H)7.93( d,J=9.17Hz,1H)8.16(d,J=8.62Hz,1H)8.20(d,J=13.39Hz,1H)8.85(d,J=13.39Hz,1H)

[0506] 13 C NMR(151MHz,CHLOROFORM-d)(ppm)9.85(s,1C)19.71(s,1C)29.37(s,1C)77.27(br.s.,1C)112.42(s,1C)114.23-114.40(m,1C)122.22 (s,1C)124.45(s,1C)128.39(s,1C)128.76(s,1C)129.00(s,1C)131.28(s,1C)133.66(s,1C)134.19(s,1C)140.00(s,1C)157.93(s,1C)

[0507] HRMS(ESI)calcd for C16H17NO3Na+[M+Na]+:294.1101,found:294.1101.

[0508] Although specific embodiments of the present invention have been described in detail, those skilled in the art can make various modifications and substitutions to the details of the technical solutions of the present invention based on all the teachings disclosed, and all such modifications and substitutions are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of the following compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment of Alzheimer's disease. 。 2. Use of a pharmaceutical composition in the preparation of a medicament for treating Alzheimer's disease, said pharmaceutical composition comprising ligand L and a compound or a pharmaceutically acceptable salt thereof, wherein, The ligand L is a ligand of a nuclear receptor that forms a dimer with the nuclear receptor RXR; The compound is .

3. The use according to claim 2, wherein the RXR dimer is a homodimer or heterodimer of RXR.

4. The use as described in claim 3, wherein the heterodimer of RXR is selected from the dimer formed by RXR and RAR, PPAR or LXR.

5. The use as described in claim 3, wherein the heterodimer of RXR is selected from RXRα / RARα, RXRα / PPARγ, and RXRα / LXRα.

6. The use as described in claim 2, wherein the ligand L is selected from ligands of RARα, ligands of PPARγ, and ligands of LXRα.

7. The use according to claim 2, wherein the ligand L is selected from all-trans retinoic acid (ATRA), rosiglitazone, and T0901317.

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