Use of derivatives based on the structure of atractylenolide and pharmaceutical compositions thereof

Abstract

The application relates to application of a smilagenin compound in preparation of a medicine for treating diseases caused by abnormal mitochondrion function, characterized in that the structural formula of the compound is shown in general formula (I). Through activity test of the smilagenin compound in related in-vivo and in-vitro models, it is found that many derivative compounds have superior cell protection activity, especially for various brain neuron cells related to mitochondrion, and the compounds have unexpected protection activity and very excellent blood-brain permeability, so that the compounds have potential wide application and great value for treating various diseases caused by abnormal mitochondrion function, make up for the deficiency of the smilagenin compound in the prior art, and have important scientific and commercial application value.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202210152310.X, filed on February 18, 2022, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of pharmaceutical technology, and particularly to the field of pharmaceutical chemical synthesis. Specifically, it refers to a novel use of a derivative based on the strychnine saponin class structure and its pharmaceutical composition in the preparation of drugs for treating diseases associated with mitochondrial dysfunction. Background Technology

[0004] Anemarrhena asphodeloides Bunge is a common traditional Chinese medicine. It is the dried tuber of the plant, belonging to the Liliaceae family. It is used to relieve thirst and internal heat, dispel pathogenic factors, and reduce edema of the limbs, and is a commonly used yin-nourishing herb. Its extracts have been shown to possess diuretic, anti-diabetic, anti-platelet aggregation, antifungal, and metabolic regulatory activities, and also exhibit inhibitory effects on cyclic adenosine monophosphate phosphodiesterase. The main chemical components of the extract include steroidal saponins, biphenylpyranones, polysaccharides, and lignins. Among these, steroidal saponins include anemarrhena saponins AI, A-II, A-III, A-IV, BI, B-II, and B-III, as well as malcosanoside 3-O-β-D-glucopyranosyl(1→2)-β-D-galactopyranoside B, degalactoside, F-glycine saponin, and isosmilax saponin. In addition, it also contains Anemarrhena asphodeloides polysaccharides A / B / C / D, cis-cylindrical resin phenol, monomethyl-cis-cylindrical resin phenol, oxidized-cis-cylindrical resin phenol, 2,6,4'-trihydroxy-4-methoxybenzophenone, p-hydroxyphenyl crotonol, vinyl pentadecanoate, β-sitosterol, mangiferin, nicotinic acid, nicotinamide and pantothenic acid, etc.

[0005] Mitochondrial function and behavior are central to human physiology. Mitochondria perform a variety of interconnected functions, producing ATP and many biosynthetic processes, while also contributing to cellular stress responses such as autophagy and apoptosis. Mitochondria form a dynamic, interconnected network, tightly bound together with other cellular compartments. Furthermore, mitochondrial functions extend beyond cell boundaries, influencing the physiology and organization of organisms by regulating intercellular communication. The mitochondrial respiratory chain is primarily composed of mitochondrial respiratory chain enzymes, and deficiencies in these enzyme complexes are a significant cause of mitochondrial diseases (approximately 30%-40% of mitochondrial diseases are due to mitochondrial respiratory chain enzyme deficiencies). The structure and function of the human respiratory chain supercomplex: oxidative phosphorylation is performed stepwise by five respiratory chain protein complexes located on the inner mitochondrial membrane. These five protein complexes are complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (ATP synthase).

[0006] Therefore, it is not surprising that mitochondrial dysfunction is a key factor in the pathogenesis of various diseases, including neurodegenerative diseases and metabolic disorders. The literature also reviews the progress of mitochondrial biology and discusses its relevance to human diseases (doi:10.1016 / j.cell.2012.02.035.). Currently, many small molecule drugs target respiratory chain complexes and act on multiple indications, providing various options for disease treatment. The number of drugs currently available targeting respiratory chain complexes for various indications includes: cancer, cardiovascular diseases, endocrine diseases, immune diseases, coronavirus infection, inflammation, metabolic diseases, neurodegenerative diseases, intestinal diseases, autism, schizophrenia, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, and chronic fatigue syndrome, among others.

[0007] Small molecules acting on mitochondria can maintain the complex in an active state and allosterically regulate its activity and electron transport efficiency. This also contributes to the overall structural stability of the complex, thereby reducing the generation of mitochondrial ROS (superoxide radicals) caused by electron leakage. Based on the regulation of complex activity, it is possible to achieve overall regulation of energy metabolism pathways within the cell and even the entire organism, as well as influence epigenetic modifications of the genome.

[0008] Stroke, commonly known as apoplexy, includes ischemic stroke (cerebral infarction) and hemorrhagic stroke (cerebral parenchymal hemorrhage, intraventricular hemorrhage, and subarachnoid hemorrhage). According to the World Health Organization, stroke is defined as damage to cerebral blood vessels and focal (or systemic) brain tissue caused by various factors, resulting in clinical symptoms lasting more than 24 hours or death. It is characterized by high morbidity, disability, recurrence, and mortality rates. Stroke is the leading cause of death among Chinese residents.

[0009] Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease, is a chronic, progressive degenerative disease affecting the upper and lower motor neurons and the muscles of the trunk, limbs, and face they innervate. It commonly manifests as progressive muscle weakness, atrophy, and fasciculations due to combined damage to both upper and lower motor neurons. Clinically, it presents as progressive skeletal muscle weakness, atrophy, fasciculations, bulbar palsy, and pyramidal tract signs. Early symptoms are mild and easily confused with other diseases; patients may only experience weakness, muscle twitching, and fatigue. Gradually, these symptoms progress to generalized muscle atrophy and difficulty swallowing, eventually leading to respiratory failure. The main theories regarding the causes of motor neuron damage are: 1. Accumulation of neurotoxic substances, such as glutamate buildup between nerve cells, eventually causing damage; 2. Free radical damage to nerve cell membranes; 3. Deficiency of nerve growth factor, preventing nerve cells from continuously growing and developing. Currently, Rilutek is the only internationally recognized drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of amyotrophic lateral sclerosis (ALS), and it is crucial to start its use as early as possible. Simultaneously, international research is exploring the use of Rilutek in combination with neurotrophic factors, antioxidants such as vitamin E and vitamin C, as well as creatine and CoQ10, for protective treatment of ALS, but this requires further confirmation through clinical trials.

[0010] Neurodegenerative diseases are divided into acute and chronic neurodegenerative diseases. Acute neurodegenerative diseases mainly include stroke, brain injury (BI), and epilepsy; chronic neurodegenerative diseases mainly include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), and Pick's disease. The causes of neurodegenerative diseases are mainly in the following four aspects: 1. Oxidative stress. Oxidative stress is caused by excessive production of free radicals and / or failure to clear them in time, resulting in an imbalance between oxidation and antioxidation in the body, leading to cell and tissue damage. Free radicals are atoms or groups with unpaired electrons, including hydroxyl radicals, superoxide anions, nitric oxide, etc. In recent years, oxidative damage to nerve tissue has been found in neurodegenerative diseases such as AD, PD, and ALS; 2. Mitochondrial dysfunction. AD patients have mtDNA defects and abnormal oxidative phosphorylation in their brains. Polymerase chain reaction (PCR) and Western blot hybridization revealed mtDNA breaks, base deletions, and mistranslation mutations in the brain tissue of sporadic AD patients. Electron microscopy confirmed an increased number of mitochondria with abnormal structure, including lamellar bodies and crystalline inclusions. Furthermore, impaired mitochondrial function in AD patients leads to insufficient neuronal energy supply and the release of large amounts of ROS, inducing oxidative stress damage, calcium imbalance, and ultimately triggering neuronal apoptosis. 3. Excitotoxicity. High glutamate concentrations in the intercellular space can produce toxins on neurons, leading to neuronal degeneration, aging, and death. This excitotoxic effect of glutamate is closely related to the occurrence and development of various neurodegenerative diseases and is one of the important mechanisms leading to neuronal death in neurodegenerative diseases. 4. Immune inflammation. Numerous studies have shown that inflammation plays a significant role in the pathogenesis of AD. The innate immune system is a natural immune defense function formed during phylogenetic development and evolution. Compared to another specific immune response, it can rapidly react to various harmful substances to protect the body. Activation of the innate immune system itself is a double-edged sword. Long-term and uncontrolled stimulation by harmful substances (such as aggregated Aβ) can trigger the innate immune system and cause damage to the brain.

[0011] COVID-19, or Coronavirus Disease 2019 for short, is an acute respiratory infectious disease caused by infection with the novel coronavirus, as named by the World Health Organization (WHO). On March 11, 2020, the WHO assessed the current COVID-19 outbreak and declared it a global pandemic. The clinical manifestations of COVID-19 pneumonia are primarily fever, fatigue, and dry cough, often accompanied by hypoxia. About half of the patients develop respiratory distress within a week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, and coagulation disorders. Currently, prevention methods for COVID-19 mainly involve vaccination and isolation. Treatment medications include Paxlovid, Azvudine tablets, Monopravir capsules, Sanhan Huashi granules, and Qingfei Paidu decoction.

[0012] The search for highly effective, selective, low-toxicity, and readily permeable novel drugs remains extremely challenging. Preventing, reducing, and treating COVID-19 infection, lowering mortality rates, and eliminating sequelae are of paramount importance. Lowering free radical levels in the brain is considered a therapeutic approach; compounds of the anemarrhenasaponin class have been shown to reduce free radical levels and protect mitochondria, enabling them to utilize oxygen more effectively.

[0013] Therefore, the development and rational use of compounds for the treatment of diseases caused by abnormalities in the mitochondrial respiratory chain are of great practical value. Summary of the Invention

[0014] The purpose of this invention is to overcome the deficiencies in the prior art. To achieve this purpose, the first aspect of this invention provides the use of a derivative based on the sarcosinate-glucanogene structure in the preparation of a medicament for treating diseases associated with mitochondrial dysfunction. Its main feature is that the derivative has the structural formula shown in general formula I.

[0015]

[0016] The derivative represented by general formula I is formed by linking the following fragments A and B.

[0017]

[0018] Where Z is NR 1 R 2 ;R 1 and R 2 Each is independently hydrogen, substituted or unsubstituted C1-C 10 Alkyl, C1-C 10The alkyl substituents are selected from halogens, hydroxyl groups, amino groups, nitro groups, cyano groups, C1-C4 alkyl groups, C1-C4 haloalkyl groups, C1-C4 alkoxy groups, C3-C6 cycloalkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, phenyl groups, benzyl groups, and C3-C6 alkyl groups. 14 Heterocyclic group, C3-C 14 Heteroaryl, wherein the heteroatom is selected from one or more of N, O, and S; or R 1 and R 2 Together they form a three- to eight-membered ring, wherein the three- to eight-membered ring has one or more elements selected from C1-C2. 10 Alkyl, C3-C 10 cycloalkyl, C6-C 20 Aryl, or C3-C 14 Heteroaryl, halogen, hydroxyl, amino, alkoxy, -CF3, -SF5, or heteroatom of sulfur, oxygen, NH, or NR a Substituents of three to eight-membered heterocycles, wherein the heteroatoms are selected from one or more of N, O, and S;

[0019] X is C(O) or S(O)2;

[0020] Y is C(R) d (R) e ), C(O) or S(O)2, R d R e Each is independently hydrogen or has at least one substituent in a C1-C group. 10 Alkyl, C3-C 10 cycloalkyl, C6-C 20 Aryl, or C3-C 14 Heteroaryl, wherein the substituents are selected from halogen, hydroxyl, amino, nitro, cyano, aldehyde, carboxyl, alkoxy, -CF3 or -SF5, and the heteroatom is selected from one or more of N, O, S, or R d and R e Together they form a three- to eight-membered ring, wherein the three- to eight-membered ring has one or more members selected from C1-C10 alkyl, C3-C10 cycloalkyl, C 6- C 20 Aryl, or C3-C 14 Heteroaryl, halogen, hydroxyl, amino, alkoxy, -CF3, -SF5, or heteroatom of sulfur, oxygen, NH, or NR a Substituents of three to eight-membered heterocycles, wherein the heteroatoms are selected from one or more of N, O, and S;

[0021] X2 is O, S, or NH;

[0022] R a Independently hydrogen or C1-C having at least one substituent 10 Alkyl, C3-C 10cycloalkyl, C6-C 20 Aryl, or C3-C 14 Heteroaryl, wherein the substituents are selected from halogen, hydroxyl, amino, nitro, cyano, alkoxy, -CF3 or -SF5, and the heteroatom is selected from one or more of N, O, and S;

[0023] n is an integer from 0 to 10 and n is not 0, m is 1, or n is 0 and m is 1, or n is an integer from 0 to 10 and n is not 0, m is 0;

[0024] R3, R 4a R 4b R 5a R 5b Each is independently hydrogen or selected from halogens, substituted alkyl groups, hydroxyl groups, and amino groups;

[0025] Indicates a single or double bond;

[0026] Each "*" independently represents a racemic, S, or R configuration.

[0027] Preferably, the structural formula of the derivative is shown in general formula (II).

[0028]

[0029] Preferably, the derivative has the structural formula shown in general formula III.

[0030]

[0031] Preferably, the structural formula of the derivative is shown in general formula IV.

[0032]

[0033] Preferably, the derivative has the structural formula shown in general formula V.

[0034]

[0035] Wherein, R6 is hydrogen, substituted or unsubstituted C1-C 10 Alkyl, C1-C 10 The alkyl substituents are selected from halogens, hydroxyl groups, -NH2, nitro groups, -CN groups, C1-C4 alkyl groups, C1-C4 haloalkyl groups, C1-C4 alkoxy groups, C3-C6 cycloalkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, phenyl groups, benzyl groups, pyridyl groups, -CO alkyl groups, -CO aromatic groups, -SO2 alkyl groups, -SO2 aromatic groups, -CO2 alkyl groups, C2-C4 (CO) alkenyl groups, -CO2 aromatic groups, and -SO3H groups.

[0036] L is hydrogen, substituted or unsubstituted C1-C 10 Alkyl, C1-C 10 The substituents of the alkyl group are selected from halogen, hydroxyl, -NH2, nitro, -CN, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C3-C6 cycloalkyl, C2-C4 alkenyl, and C2-C4 alkynyl.

[0037] n is an integer from 0 to 10;

[0038] n2 is 0, 1, 2, or 3;

[0039] m and m' are independent integers from 1 to 4;

[0040] W1 is either C or NH;

[0041] V1 is either C or NH;

[0042] M is C, S, O, or NH;

[0043] Preferably, the structural formula of the derivative is shown in general formula VI.

[0044]

[0045] Y1 is C(Rd)(Re), C(O) or S(O)2, where Rd and Re are independently hydrogen or C1-C having at least one substituent. 10 Alkyl, C3-C 10 cycloalkyl, C6-C 20 Aryl, or C3-C 14 Heteroaryl, wherein the substituents are selected from halogens, hydroxyl groups, amino groups, nitro groups, cyano groups, aldehyde groups, carboxyl groups, alkoxy groups, -CF3 or -SF5 groups, and the heteroatom is selected from one or more of N, O, and S, or R d and R e Together they form a three- to eight-membered ring, wherein the three- to eight-membered ring has one or more elements selected from C1-C2. 10 Alkyl, C3-C 10 cycloalkyl, C6-C 20 Aryl, or C3-C 14 Heteroaryl, halogen, hydroxyl, amino, alkoxy, -CF3, -SF5, or heteroatom of sulfur, oxygen, NH, or NR a The substituents and heteroatoms of the three- to eight-membered heterocycles are selected from one or more of N, O, and S;

[0046] L is hydrogen, substituted or unsubstituted C1-C 10 Alkyl, C1-C 10The substituents of the alkyl group are selected from halogen, hydroxyl, -NH2, nitro, -CN, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C3-C6 cycloalkyl, C2-C4 alkenyl, and C2-C4 alkynyl.

[0047] n is an integer from 0 to 10;

[0048] n2 is 0, 1, 2, or 3;

[0049] n3 is an integer from 1 to 10.

[0050] m is an integer from 0 to 10.

[0051] Preferably, the structural formula of the derivative is shown in general formula VII.

[0052]

[0053] Among them, R6 and R7 are independently hydrogen, substituted or unsubstituted C1-C. 10 Alkyl, C1-C 10 The substituents of the alkyl group are selected from halogen, hydroxyl, -NH2, nitro, -CN, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C3-C6 cycloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, phenyl, benzyl, pyridyl, -CO alkyl, -CO aromatic, -SO2 alkyl, -SO2 aromatic, -CO2 alkyl, C2-C4 (CO) alkenyl, -CO2 aromatic, -SO3H;

[0054] L is hydrogen, substituted or unsubstituted C1-C 10 Alkyl, C1-C 10 The substituents of the alkyl group are selected from halogen, hydroxyl, -NH2, nitro, -CN, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C3-C6 cycloalkyl, C2-C4 alkenyl, and C2-C4 alkynyl.

[0055] W2 is either C or NH;

[0056] V2 is C, O, S, or NH;

[0057] n is an integer from 0 to 10;

[0058] n1 is an integer from 1 to 10;

[0059] n2 can be 0, 1, 2, or 3.

[0060] Preferably, the structural formula of the derivative is shown in general formula VIII.

[0061]

[0062] Z1 is hydrogen, substituted or unsubstituted C1-C. 10 Alkyl, C1-C 10 The alkyl substituents are selected from halogens, hydroxyl groups, -NH2, nitro groups, -CN groups, C1-C4 alkyl groups, C1-C4 haloalkyl groups, C1-C4 alkoxy groups, C3-C6 cycloalkyl groups, C2-C4 alkenyl groups, C2-C4 alkynyl groups, phenyl groups, benzyl groups, pyridyl groups, -CO alkyl groups, -CO aromatic groups, -SO2 alkyl groups, -SO2 aromatic groups, -CO2 alkyl groups, C2-C4 (CO) alkenyl groups, -CO2 aromatic groups, and -SO3H groups.

[0063] W3 can be C, S, O, or NH;

[0064] n is an integer from 0 to 10;

[0065] n4, n5, n6, and n7 are integers from 1 to 4.

[0066] In another preferred embodiment, fragment B in the structural formula of the derivative has the following structure:

[0067]

[0068] Preferably, the derivative is one of the following compounds, a mixture of diastereomers of the following compounds, or an enantiomer of the following compounds.

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] Preferably, the derivatives include corresponding deuterated compounds resulting from the substitution of any one or more hydrogen atoms thereon with its stable isotope deuterium.

[0105] In another aspect, the present invention provides a pharmaceutical composition comprising: a compound of general formula I described above, a pharmaceutically acceptable salt thereof, a stereoisomer, a tautomer thereof, a prodrug, or a pharmaceutically acceptable carrier thereof.

[0106] Preferably, it also includes an adjunct therapy, which includes an antidepressant, an antimanic drug, a Parkinson's disease treatment drug, an Alzheimer's disease treatment drug, or a combination thereof.

[0107] Preferably, the pharmaceutically acceptable salt is selected from the group consisting of: hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, trifluoromethanesulfonate, benzenesulfonate, p-toluenesulfonate (toluenesulfonate), 1-naphthalenesulfonate, 2-naphthalenesulfonate, acetate, trifluoroacetate, malate, tartrate, citrate, lactate, oxalate, succinate, fumarate, maleate, benzoate, salicylate, phenylacetate, and mandelate.

[0108] Preferably, the additional therapeutic agent is moclobemide, toloxacin, fluoxetine, paroxetine, citalopram, sertraline, venlafaxine, trimethoprim, trazodone, imipramine, desipramine, clomipramine, amitriptyline, nortriptyline, doxepin, maprotiline, loxapine, amoxapine, mirtazapine, buspirone, clomezadone, tandospirone, lithium carbonate, tacrine, huperzine A, galantamine, donepezil, lifanstigmine, memantine, pramipexole, talicrazole, ropirocin, or a combination thereof.

[0109] The present invention also provides the use of the pharmaceutical composition in the preparation of medicaments for the protection, treatment, therapy or relief of a patient’s disease, ailment or condition.

[0110] Preferably, the related diseases, symptoms, or conditions caused by mitochondrial abnormalities are specifically related diseases, symptoms, or conditions caused by respiratory chain abnormalities, including four major categories: metabolic diseases, tumors, inflammation, and central nervous system diseases.

[0111] Metabolic diseases include: hyperglycemia, hyperlipidemia, hypercholesterolemia, high LDL cholesterol, low HDL cholesterol, angiogenic disorders, non-alcoholic fatty liver disease, cerebrovascular accident, myocardial infarction, atherosclerosis, coronary heart disease, anti-aging, urinary urgency and frequency, type I diabetes, chronic obstructive pulmonary disease, etc.

[0112] Tumors include: benign prostatic hyperplasia, Wegener's granulomatosis, pulmonary sarcoidosis, leukemia, lymphoma, pancreatic cancer, neurotumor, etc.

[0113] Inflammation includes: peripheral neuritis, chemotherapy-induced peripheral neuritis, autoimmune diseases, organ transplant-related symptoms, influenza virus, coronavirus (prevention, treatment, and sequelae elimination of infection), acute respiratory distress syndrome, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, retinal detachment, retinitis pigmentosa, macular degeneration, pancreatitis, atopic dermatitis, rheumatoid arthritis, spondyloarthritis, gout, systemic lupus erythematosus, Sjögren's syndrome, systemic scleroderma, antiphospholipid syndrome, vasculitis, osteoarthritis, autoimmune hepatitis, autoimmune hepatobiliary diseases, primary sclerocholangitis, nephritis, celiac disease, autoimmune ITP, transplant rejection, ischemia-reperfusion injury of solid organs, sepsis, periodontitis, systemic inflammatory response syndrome, myocarditis, allergic diseases, asthma, interleukin-1 converting enzyme-related fever syndrome, Behcet's disease, etc.

[0114] Central nervous system diseases include: Pick's disease, spinal cord injury repair, depression, anxiety, Parkinson's disease, Alzheimer's disease, sleep disorders, ischemic stroke, hemorrhagic stroke, amyotrophic lateral sclerosis, traumatic brain injury, brain atrophy, Huntington's disease, schizophrenia, mania, drug withdrawal, multiple sclerosis, sleep improvement, myasthenia gravis, etc.

[0115] This invention utilizes compounds, pharmaceutical compositions, and their applications based on sarsaponin compounds. Through in vitro and in vivo model activity tests on sarsaponin compounds, it was unexpectedly discovered that many derivative compounds possess superior cytoprotective activity, particularly exhibiting surprising protective activity against various brain neurons. Furthermore, these compounds demonstrate excellent blood-brain permeability, suggesting a wide potential range of applications and significant value in treating various diseases caused by mitochondrial dysfunction. This invention addresses the shortcomings of existing technologies in the application of sarsaponin compounds and possesses significant scientific and commercial application value. Attached Figure Description

[0116] Figure 1 The effects of different small molecule compounds on neuronal cell death.

[0117] Figure 2 This study investigated the oxidative damage induced by hydrogen peroxide (H2O2) in human SHSY5Y neurotumor cells by small molecule compounds.

[0118] Figure 3 This represents the proportion of dead neurons.

[0119] Figure 4 Phenotypic diagrams showing the effects of each experimental group on inflammation in zebrafish.

[0120] Figure 5 The effect of each experimental group on inflammation (neutrophil count) in zebrafish.

[0121] Figure 6 The anti-inflammatory effects of each experimental group on inflammatory zebrafish.

[0122] Figure 7 Changes in cerebral blood flow in mice during surgery.

[0123] Figure 8 The changes in body weight of mice before and after surgery.

[0124] Figure 9 Changes in grip strength in the forelimbs of mice.

[0125] Figure 10 The score for neurological deficits in mice.

[0126] Figure 11 The volume of cerebral infarction in mice.

[0127] Figure 12 The volume of brain edema in mice.

[0128] Figure 13 The immobility time of the mouse FST.

[0129] Figure 14 The sugar water preference rate in mouse SPT.

[0130] Figure 15 The immobility time (TST) in mice is 1.

[0131] Figure 16 To detect ROS levels in mouse serum.

[0132] Figure 17 To detect the concentration of H2O2 in mouse serum.

[0133] Figure 18 To detect NO concentration in mouse serum.

[0134] Figure 19 To detect lipid peroxidation levels in mouse serum.

[0135] Figure 20 To detect ROS levels in the hippocampus of mice.

[0136] Figure 21 To detect the concentration of H2O2 in the hippocampus of mice.

[0137] Figure 22 To detect NO concentration in the hippocampus of mice.

[0138] Figure 23 To detect lipid peroxidation levels in the hippocampus of mice.

[0139] Figure 24 To detect the concentration of IL-1β in mouse serum.

[0140] Figure 25 To detect the concentration of IL-6 in mouse serum.

[0141] Figure 26 To detect the concentration of IL-10 in mouse serum.

[0142] Figure 27 To detect the concentration of IL-1β in the hippocampus of mice.

[0143] Figure 28 To detect the concentration of IL-6 in the hippocampus of mice.

[0144] Figure 29 To detect the concentration of IL-10 in mouse serum.

[0145] Figures 30a to 32b These represent the binding strengths of each small molecule to respiratory chain complex I.

[0146] Figure 33 The graph shows the activity results of each small molecule on SMP.

[0147] Figure 34 This is a schematic diagram showing the effect of various small molecules on cellular oxygen consumption.

[0148] Figures 35a to 35b The figures show the results of small molecules on mitochondrial ROS and mitochondrial transmembrane potential difference, respectively.

[0149] Figures 36a to 36d The images show the results of small molecules on APOE-induced atherosclerosis in mice.

[0150] Figures 37a to 37g The image shows the results of the small molecule on the water maze test in AD rats.

[0151] Figure 38 This is a diagram showing the results of small molecule stimulation in the T-maze test of AD rats.

[0152] Figure 39a and 39b The figure shows the results of small molecules on nesting behavior in AD mice.

[0153] Figure 40a and 40b The image shows the results of the small molecule on the water maze test in AD mice.

[0154] Figure 41 This is a diagram showing the results of light and dark chamber experiments on small molecules in AD mice.

[0155] Figure 42a and 42b For small molecules of TDP43 A315T The results of mouse survival time are shown in the figure.

[0156] Figure 43 For small molecules of SODG93A Figure showing the results of mouse gait analysis.

[0157] Figure 44 For small molecules of SOD G93A Results of open field experiment in mice.

[0158] Figure 45a and 45b The figure shows the results of small molecules acting on the DSS mouse enteritis model.

[0159] Figure 46a and 46b This is a diagram showing the results of small molecules acting on a rat model of TNBS enteritis.

[0160] Figure 47 The graph shows the effect of small molecules on blood glucose levels in DB mice.

[0161] Figures 48a to 48d The graph shows the results of body weight and body fat percentage in DIO mice.

[0162] Figure 49 This is a diagram showing the in vivo killing effect of small molecules on hematologic malignancies.

[0163] Figure 50 The diagram shows the protective effect of small molecules on substantia nigra neurons.

[0164] Figure 51 This is a diagram showing the protective effect of small molecules against viral infection.

[0165] Figure 52 This is a diagram showing the results of small molecules in a rat model of acute traumatic brain injury (TBI).

[0166] Figure 53 This figure shows the in vitro killing effect of small molecules on pancreatic cancer cells.

[0167] Figure 54 This is a diagram showing the effect of small molecules on excessive urination in DB animals.

[0168] Figures 55a to 55c The figure shows the results of small molecules affecting cardiovascular inflammation in APOE mutant mice fed a high-fat diet.

[0169] Figure 56a and 56b The figure shows the results of a pharmacological experiment on the effect of small molecules on subthreshold hypnotic doses of sodium pentobarbital on sleep in mice. Detailed Implementation

[0170] To better understand the technical content of this invention, the specific implementation method of this invention will be further described below.

[0171] In this invention, the term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, including straight-chain and branched hydrocarbon groups, such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), tert-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0172] In this invention, the term "alkyl" includes substituted or unsubstituted alkyl groups.

[0173] In this invention, the term "substituted or unsubstituted" means that the group may be unsubstituted, or that the H in the group is substituted by one or more (preferably 1 to 6, more preferably 1 to 3) substituents.

[0174] In this invention, "substituted" means that the group has one or more (preferably 1 to 6, more preferably 1 to 3) substituents selected from the group consisting of: halogen, hydroxyl, -NH2, nitro, -CN, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C3-C6 cycloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, phenyl, benzyl, C2-C8 heterocyclic, C2-C8 heteroaryl, and the heteroatom is selected from one or more of N, O and S.

[0175] In this invention, the term "cycloalkyl" refers to a substituted or unsubstituted C3-C alkyl group. 12 Cycloalkyl.

[0176] In this invention, the term "alkoxy" refers to an -O-alkyl group, wherein the alkyl group may be saturated or unsaturated, and may be branched, linear, or cyclic. Preferably, the alkoxy group has 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Representative examples include (but are not limited to): methoxy, ethoxy, and propoxy.

[0177] In this invention, the term "aryl" refers to a monovalent aromatic carbocyclic group with 6 to 20 (preferably 6 to 14) carbon atoms, having a monocyclic (e.g., phenyl) or fused ring (e.g., naphthyl or anthracene). If the bonding point is on an aromatic carbon atom, the fused ring may be non-aromatic (e.g., 2-benzoxazolone, 2H-1,4-benzoxazine-3(4H)-one-7-yl, etc.). Preferred aryl groups include phenyl and naphthyl. The term includes substituted or unsubstituted forms, wherein the substituents are defined as above.

[0178] In this invention, the term "alkenyl" refers to an alkenyl group having 2 to 10 (e.g., 2 to 6 or 2 to 4) carbon atoms and having at least 1 (e.g., 1 to 2) unsaturated alkene bonds (>C=C<). Examples of such groups include vinyl, allyl, and but-3-alkenyl.

[0179] In this invention, the term "cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms and possessing a monocyclic or polycyclic structure (including fused, bridged, and spirocyclic systems). In fused-ring systems, one or more rings may be cycloalkyl, heterocyclic, aryl, or heteroaryl, as long as the linking site is a ring through a cycloalkyl group. Suitable examples of cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl.

[0180] In this invention, the term "halogenated" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0181] In this invention, the term "heteroaryl" refers to an aromatic group having 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulfur within a ring. Such a heteroaryl can be monocyclic (e.g., pyridyl or furanyl) or fused-ring (e.g., indolizinyl or benzothiophene), wherein the fused ring can be non-aromatic and / or contain one heteroatom, provided the connecting point is through an atom of an aromatic heteroaryl group. In one embodiment, the nitrogen and / or sulfur atom of the heteroaryl ring is optionally oxidized to an N-oxide (NO), sulfinyl, or sulfonyl group. Preferably, heteroaryls include pyridyl, pyrroleyl, indolyl, thiophene, and furanyl. This term includes substituted or unsubstituted heteroaryls.

[0182] In this invention, the term "substituted heteroaryl" refers to a heteroaryl group substituted by 1 to 5, preferably 1 to 3, more preferably 1 to 2 substituents selected from the same substituents as defined for substituted aryl.

[0183] In this invention, the terms "heterocyclic," "heterocyclic," "heterocyclic alkyl," or "heterocyclic group" refer to a saturated, partially saturated, or unsaturated group (but not aromatic) having a monocyclic or fused ring (including bridged and spirocyclic systems) containing 1 to 10 carbon atoms and 1 to 4 (e.g., 3) heteroatoms selected from nitrogen, sulfur, or oxygen. In fused ring systems, one or more rings may be cycloalkyl, aryl, or heteroaryl, provided the junction passes through a non-aromatic ring. In one embodiment, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, a sulfinyl group, and a sulfonyl moiety.

[0184] In this invention, the terms "substituted heterocyclic" or "substituted heterocyclic alkyl" or "substituted heterocyclic group" refer to a heterocyclic group substituted by 1 to 5 (e.g., 1 to 3) substituents, which are the same as the substituents defined by substituted cycloalkyl.

[0185] In this invention, the term "stereoisomer" refers to a compound with one or more stereocenters that have different chiralities. Stereoisomers include enantiomers and diastereomers.

[0186] In this invention, the term "tautomer" refers to an alternative form of a compound with different proton positions, such as enol-ketone and imine-enamine tautomers, or a heteroaryl tautomer containing a ring atom connected to both the -NH- and N-parts of the ring, such as pyrazole, imidazole, benzimidazole, triazole, and tetraazole.

[0187] The present invention provides a pharmaceutical composition comprising an active ingredient within a safe and effective range, and a pharmaceutically acceptable carrier.

[0188] The "active ingredient" as described in this invention refers to the compound of general formula (I) described in this invention or its pharmaceutically acceptable salt, its stereoisomer or its tautomer, or its prodrug.

[0189] The "active ingredient" and pharmaceutical composition described in this invention can be used as mitochondrial protectants. In another preferred embodiment, it is used to prepare a medicament for the prevention and / or treatment of neurodegenerative diseases. In another preferred embodiment, it is used to prepare a medicament for the prevention and / or treatment of mitochondrial-related metabolic diseases.

[0190] "Safe and effective dose" refers to an amount of active ingredient sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1–2000 mg of active ingredient per dose, more preferably 10–200 mg of active ingredient per dose. Preferably, "one dose" refers to one tablet.

[0191] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the active ingredient of the present invention without significantly reducing the efficacy of the active ingredient.

[0192] The compounds of the preferred embodiments of the present invention can be administered as a single active agent or in combination with one or more other agents for treating cancer.

[0193] Typically, the compounds of preferred embodiments are administered in a therapeutically effective amount, via any acceptable mode of administration of an agent with similar effects. The actual dosage of the compound (i.e., the active ingredient) of preferred embodiments is determined based on several factors, such as the severity of the disease to be treated, the patient's age and relative health, the potency of the compound used, the route and form of administration, and other factors. The drug may be administered multiple times a day, preferably once or twice daily. All these factors are taken into consideration by the attending physician.

[0194] For the purposes of preferred embodiments, the therapeutically effective dose can typically be a total daily dose administered to the patient in a single dose or in divided doses, for example, from about 0.001 to about 1000 mg / kg body weight daily, preferably from about 1.0 to about 30 mg / kg body weight daily. A dosage unit composition may include its dose factor to form a daily dose. The choice of dosage form depends on various factors, such as the administration method and the bioavailability of the pharmaceutical substance. Generally, the compounds of preferred embodiments can be administered as pharmaceutical compositions via any route suitable for the condition being treated. Suitable routes include, but are not limited to, oral, parenteral (including subcutaneous, intramuscular, intravenous, intra-arterial, intradermal), vaginal, intraperitoneal, intrapulmonary, and intranasal routes. It should be understood that preferred routes may vary depending on the patient's condition. A preferred route of administration is oral, with a convenient daily dose that can be adjusted according to the degree of bitterness. It can be formulated with pharmaceutically acceptable carriers or excipients into tablets, pills, capsules, semi-solids, powders, sustained-release formulations, solutions, suspensions, elixirs, aerosols, or any other suitable composition, etc. When the compound is formulated for parenteral administration, it can be formulated with a pharmaceutically acceptable parenteral carrier. Another preferred method of administration of the preferred embodiment compound is inhalation. This is an effective method of delivering a therapeutic agent directly to the respiratory tract (see, for example, U.S. Patent No. 5,607,915).

[0195] This invention allows for the administration of compounds in any convenient formulation. The term "formulation" as used herein refers to a dosage form that facilitates drug delivery, containing a compound of general formula I, such as, but not limited to, aqueous injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder inhalers, sustained-release formulations, and controlled-release formulations. These pharmaceutical excipients can be those conventionally used in various formulations, such as, but not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubricants; or they can be selected for compatibility with the substance, such as emulsifiers, solubilizers, antibacterial agents, analgesics, and antioxidants. These excipients can effectively improve the stability and solubility of the compounds contained in the composition or alter the release and absorption rates of the compounds, thereby improving the metabolism of the compounds in vivo and enhancing the drug delivery effect. In addition, excipients may be used to achieve specific drug delivery purposes or methods, such as sustained-release, controlled-release, and pulsatile drug delivery. These excipients include, but are not limited to, gelatin, albumin, chitosan, polyethers, and polyester polymers, such as, but not limited to, polyethylene glycol, polyurethane, polycarbonate, and their copolymers. The term "beneficial" primarily manifests in, but is not limited to, improved therapeutic efficacy, increased bioavailability, reduced toxicity and side effects, and improved patient compliance.

[0196] Suitable pharmaceutically acceptable carriers or excipients include: processing agents and drug delivery modifiers and accelerators, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, glucose, hydroxypropyl-β-cyclodextrin, sodium sulfobutyl-β-cyclodextrin, polyvinylpyrrolidone, low-melting-point waxes, ion exchange resins, etc., and any combination of two or more thereof. Liquid and semi-solid excipients may be selected from glycerol, propylene glycol, water, ethanol, and various oils, including petroleum, animal, vegetable, or synthetic sources such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous glucose solutions, and ethylene glycol. Other suitable pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991), and are incorporated herein by reference.

[0197] In this invention, the term "pharmaceutically acceptable salt" refers to a nontoxic acid or alkaline earth metal salt of a compound of formula I. These salts can be prepared in situ during the final isolation and purification of the compound of formula I, or by reacting a suitable organic or inorganic acid or base with a basic or acidic functional group. Representative salts include, but are not limited to: acetates, adipates, alginates, citrates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, butates, camphorates, camphorsulfonates, diglucuronates, cyclopentanepropionates, dodecyl sulfates, ethanesulfonates, glucono-heptates, glycerol phosphates, hemisulfates, heptanates, hexanoates, fumarates, hydrochlorides, hydrobromides, hydroiodates, 2-hydroxyethanesulfonates, lactates, maleates, methanesulfonates, nicotinates, 2-naphthylsulfonates, oxalates, dihydroxynaphthyl salts, pectates, thiocyanates, 3-phenylpropionates, picrates, neopentanoates, propionates, succinates, sulfates, tartrates, thiocyanates, salicylates, p-toluenesulfonates, and undecanoates. Furthermore, nitrogen-containing basic groups can be quaternized with reagents such as: alkyl halides, such as chlorides, bromides, and iodides of methyl, ethyl, propyl, and butyl groups; dialkyl sulfates, such as dimethyl, diethyl, dibutyl, and dipentyl sulfates; long-chain halides, such as chlorides, bromides, and iodides of decyl, lauryl, myristyl, and stearyl groups; and aralkyl halides, such as benzyl and phenethyl bromides. This yields water-soluble, oil-soluble, or dispersible products. Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, methanesulfonic acid, succinic acid, and citric acid. Base addition salts can be prepared in situ during the final separation and purification of compounds of general formula I, or by reacting the carboxylic acid moiety with a suitable base (such as a pharmaceutically acceptable metal cation hydroxide, carbonate, or bicarbonate) or ammonia, or an organic primary, secondary, or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, alkali metal and alkaline earth metal-based cations, such as salts of sodium, lithium, potassium, calcium, magnesium, and aluminum, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. Other representative organic amines used to form base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, and piperazine.

[0198] In this invention, the term "pharmaceutically acceptable prodrug" refers to the prodrug of the compounds of the preferred embodiments, which are rapidly converted in vivo into the parent compound represented by the above general formula, for example, by hydrolysis in the blood. Full discussion is provided in "T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, ACS 15 Symposium Series, Vol. 14" and "Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987," both of which are incorporated herein by reference.

[0199] This invention provides a method for preparing compounds of general formula (I). Taking anemarrhenasaponin as an example, the preparation method of the key intermediate is as follows:

[0200] α configuration:

[0201] Method 1:

[0202]

[0203] Method 2:

[0204]

[0205] β configuration:

[0206] Method 1:

[0207]

[0208] Method 2:

[0209]

[0210] Mixed configuration:

[0211]

[0212] The preparation of compounds of general formula (I) is as follows: Taking the α-configuration of the anemarrhenasaponin core as an example (for other configurations or a certain configuration of other cores, the preparation method is the same as the provided method, and the synthetic route is as follows:

[0213] Option 1:

[0214]

[0215] Option 2:

[0216]

[0217] Option 3:

[0218]

[0219] In each formula, R 1 R 2 The definitions of Y and n are shown above.

[0220] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0221] The following abbreviations have the following meanings:

[0222] DBU refers to 1,8-diazabicyclo[5.4.0]undec-7-ene; DIBAL represents diisobutylaluminum hydride; DIAD refers to diisopropyl azodicarboxylate; DIEA refers to diisopropylethylamine; DMAP refers to N,N-dimethylaminopyridine; DME refers to 1,2-dimethoxyethane; DMF refers to N,N-dimethylformamide; DMPE refers to 1,2-bis(dimethylphosphino)ethane; DMSO refers to dimethyl sulfoxide; DPPB refers to 1,4-bis(diphenylphosphino)butane; DPPE refers to 1,2-bis(diphenylphosphino)ethane; DPPF refers to 1,1'-bis(diphenylphosphino)ferrocene; DPPM refers to 1,1'-bis(diphenylphosphino)methane; EDC represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HATU represents 2-(7-aza-1H-benzotriazole- 1-(1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate; HMPA indicates hexamethylphosphamide; IPA refers to isopropanol; LDA refers to lithium diisopropylamino; LHMDS refers to lithium di(trimethylsilyl)amino; LAH refers to lithium aluminum hydride; PyBOP refers to benzotriazol-1-yl-oxytripyrrolylphosphobenzotriazol hexafluorophosphate; TDA-I refers to tris(2-(2-methoxyethoxy)ethyl)amine; DCM refers to dichloromethane; TEA refers to triethylamine; TFA refers to trifluoroacetic acid; THF refers to tetrahydrofuran; NCS refers to N-chlorosuccinimide; NMM refers to N-methylmorpholine; NMP refers to N-methylpyrrolidone; PPh3 refers to triphenylphosphine; T3P refers to 1-propylphosphonic anhydride; PMA refers to phosphomolybdic acid; PE refers to petroleum ether; EA refers to ethyl acetate; RBF refers to a round-bottom flask; rt refers to room temperature.

[0223] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0224] Example 1

[0225]

[0226] The preparation process is as follows:

[0227] Step 1: Intermediate 1

[0228]

[0229] 10 g of raw material A was added to a 500 mL round-bottom flask and dissolved in 100 mL of DCM. 12.8 g of Desmartin was added at 0 °C, and after 15 min, the mixture was stirred at rt for 1 h. The reaction was monitored by TLC until completion (PMA staining). After the reaction was complete, the residue was removed by filtration. The filtrate was evaporated to dryness, redissolved in DCM, washed twice with water, dried, and purified by silica gel column chromatography to obtain 9 g of a white solid product (intermediate 1).

[0230] Step 2: Intermediate 2

[0231]

[0232] Add 5 g of intermediate 1 and 1.7 g of NH₂OH·HCl to a 100 mL round-bottom flask, dissolve in 50 mL of dry LPyridine, stir at 70 °C for 1–2 h, and monitor the reaction by TLC until completion (PMA colorimetric analysis). After the reaction is complete, remove the solvent by rotary evaporation, redissolve in DCM, wash twice with 1 N HCl aqueous solution, dry, and precipitate by silica gel column chromatography to obtain 5.2 g of pale yellow solid crude product.

[0233] Step 3: Intermediate 3

[0234]

[0235] Add 5 g of intermediate 2 to a 500 mL round-bottom flask and dissolve it in 100 mL of dry MeOH. Add 4.2 g of NiCl2·6H2O at 0 °C. After 15 min, add 2.7 g of NaBH4 in portions. After 30 min, raise the temperature to rt and stir for 4 h. The reaction was monitored by TLC until it ended (PMA colorimetric analysis). After the reaction was complete, filter to remove the residue. After evaporating the filtrate to dryness, 5 g of white solid product was obtained.

[0236] Step 4: Example 1

[0237]

[0238] In a round-bottom flask, 0.055 g of intermediate 3, 0.041 g of 3-(4-methylpiperazin-1-yl)-propionic acid, 0.046 g of EDC, 0.084 mL of triethylamine, and 0.003 g of DMAP were dissolved in 2 mL of dichloromethane and reacted at room temperature for 2 h. The reaction was then monitored. After the reaction was complete, the mixture was washed twice with saturated NH4Cl aqueous solution, dried, and purified by silica gel column chromatography to obtain 25 mg of the product.

[0239] The NMR data are as follows: 1 ¹H NMR (400MHz, CDCl₃) δ 0.76 (s, 3H), 0.80–2.30 (m, 36H), 2.25–2.80 (m, 11H), 3.25–4.10 (m, 2H), 4.150–4.55 (m, 2H), 8.60–8.70 (m, 5H); Mass spectrometry: [M+1] 570.5.

[0240] Example 1A

[0241]

[0242] The preparation process of Example 1A is as follows:

[0243] Step 1: Intermediate 4

[0244]

[0245] Under argon protection, 0.5 g of anemarrhenasaponin A, 0.4 g of p-nitrobenzoic acid, and 0.63 g of PPh3 were dissolved in 5 mL of dry THF. The mixture was stirred in an ice-water bath for 5 min, then DIAD was slowly added dropwise, and the mixture was stirred for 10 min. The ice bath was removed, and the reaction was allowed to proceed at room temperature for 3 h. The reaction was then monitored. After the reaction was complete, the solvent was evaporated, and the mixture was extracted with sodium bicarbonate solution / dichloromethane. The solution was purified by silica gel column chromatography (PE:EA = 45:1), and the result was detected by TLC (PE:EA = 15:1), with vanillin showing a positive color. The yield was 55%.

[0246] Step 2: Intermediate 5

[0247]

[0248] Add 15 mL of MeOH to 0.36 g of intermediate 4 and 0.35 g of K₂CO₃, and stir overnight at 55 °C. Dry the solvent by rotary evaporation, extract with water / dichloromethane, and dry the organic layer to obtain the product. Add directly to the next reaction step; vanillin is used for color development. Yield: 80%.

[0249] Step 3: Intermediate 6

[0250]

[0251] Under argon protection, 0.2 g of intermediate 5, 0.14 g of phthalimide, and 0.25 g of PPh3 were dissolved in 4 mL of dry THF. The mixture was stirred in an ice-water bath for 5 min, then 0.19 g of DIAD was slowly added dropwise, and the mixture was stirred for 10 min. The ice bath was removed, and the reaction was allowed to proceed at room temperature for 3 h. The reaction was then detected. The solvent was evaporated, and the mixture was extracted with water / dichloromethane. The solution was purified by silica gel column chromatography (PE:EA = 45:1), and by TLC (PE:EA = 15:1). The reaction was performed with vanillin, and the yield was 50%.

[0252] Step 4: Intermediate 7

[0253]

[0254] 10 mL of MeOH was added to 0.13 g of intermediate 6 and 0.072 g of N2H4·H2O, and the mixture was stirred overnight at 55 °C. The solvent was evaporated, the mixture was washed with water, extracted with dichloromethane, and the organic layer was evaporated to dryness to obtain the product. The product was directly added to the next reaction, and vanillin was used for color development. The yield was 90%.

[0255] Step 5: Example 1A

[0256]

[0257] Under argon protection, 0.07 g of the intermediate, 0.058 g of 3-(4-methylpiperazin-1-yl)-propionic acid, 0.065 g of EDC, and 0.01 g of DMAP were dissolved in 4 mL of dichloromethane and reacted at room temperature for 4 h. The reaction was then monitored. Extraction was performed using sodium bicarbonate solution / dichloromethane and passed through an alkaline alumina column (PE:EA = 1:1). Vanillin was used for color development, and the yield was 65%.

[0258] The NMR data are as follows: 1 H NMR (400MHz, CDCl3) δ8.85(d,J=6.9Hz,1H),4.41(dd,J=13.0,6.6Hz,1H),4.18(s,1H),3.95(d,J=10.6Hz,1H),3.30(d,J=11.6Hz,1H),3.01 -2.20(brs,8H),2.62(d,J=3.0Hz,2H),2.39(s,2H),2.30(s,3H),1.23-2.07(m,27H),1.08(d,J=6.7Hz,3H),1.05–0.93(m,6H),0.76(s,3H). Mass spectrum: [M+1]570.5.

[0259] Example 1B

[0260]

[0261] The preparation process of Example 1B is as follows:

[0262] Step 1: Intermediate 8

[0263]

[0264] Starting with raw material A, intermediate 8 was obtained under the same conditions as in step 3 of Example 1A, with a yield of 50%.

[0265] Step 2: Intermediate 9

[0266]

[0267] In this step, intermediate 9 is obtained using intermediate 8 as raw material under the same conditions as in step 4 of Example 1A, with a yield of 90%.

[0268] Step 3: Example 1B

[0269]

[0270] In this step, using intermediate 9 and 3-(4-methylpiperazin-1-yl)-propionic acid as raw materials, Example 1B was obtained under the same conditions as in step 5 of Example 1A, with a yield of 65%.

[0271] The NMR data are as follows: 1 H NMR (400MHz, CDCl3) δ8.15(d,J=7.6Hz,1H),4.33(dd,J=14.8,7.5Hz,1H),3.88(dd,J=10.9,2.4Hz,1H),3.65(m,1H),3.23(d,J=10.9Hz,1H),2. 90-2.10(brs,8H),2.56(t,J=6.2Hz,2H),2.26(m,2H),2.24(s,3H),1.2 3-2.07(m,27H),1.01(d,J=7.1Hz,3H),0.95-0.83(m,6H),0.69(s,3H). Mass spectrum: [M+1]570.5.

[0272] Example 2

[0273]

[0274] Using intermediate 3 and 1-methylpiperidine-4-carboxylic acid as raw materials, Example 2 was obtained under the same conditions as the experimental steps described in step 4 of Example 1.

[0275] The NMR data are as follows: 1HNMR (CDCl3, 400MHz): δ 0.75 (s, 3H), 0.80–2.70 (m, 43H), 2.95–4.00 (m, 7H), 4.40–4.55 (m, 1H), 6.05 (s, 1H); Mass spectrometry: [M+1] 541.5.

[0276] Example 3

[0277]

[0278] Using intermediate 3 and N,N-dimethylglycine as raw materials, Example 3 was obtained under the same conditions as the experimental steps described in step 4 of Example 1.

[0279] The NMR data are as follows: 1 HNMR (CDCl3, 400MHz): δ 0.76 (s, 3H), 0.80–2.20 (m, 36H), 2.29 (s, 3H), 2.31 (s, 3H), 2.80–3.00 (m, 2H), 3.20–3.35 (s, 1H), 3.85–4.40 (m, 3H), 7.10–7.40 (m, 1H); Mass spectrometry: [M+1] 50 1.3.

[0280] Example 4

[0281]

[0282] Using intermediate 3 and N,N-dimethyl-β-alanine as raw materials, Example 4 was obtained under the same conditions as the experimental steps described in step 4 of Example 1.

[0283] The NMR data are as follows: 1 HNMR(CDCl3,400MHz): δ0.76(s,3H),0.80-2.20(m,36H),2.32(s,3H),2.36(s,3 H),2.45-2.70(m,4H),3.25-3.40(m,1H),3.80-4.45(m,3H),8.90-9.10(m,1H). Mass spectrum: [M+1]515.3.

[0284] Example 4A

[0285]

[0286] Using intermediate 7 and N,N-dimethyl-β-alanine as raw materials, Example 4A was obtained under the same conditions as the experimental steps described in step 5 of Example 1A.

[0287] The NMR data are as follows: 1H NMR (400MHz, CDCl3) δ9.20(d,J=7.8Hz,1H),4.41(dd,J=14.1,7.6Hz,1H),4.19(d,J=7.2Hz,1H),3.95(dd,J=11.0,2.5Hz,1H),3.30(d,J=1 0.9Hz,1H),2.59–2.50(m,2H),2.38–2.33(m,2H),2.30(s,6H),1.23-2.07(m,27H),1.08(d,J=7.1Hz,3H),1.02–0.92(m,6H),0.76(s,3H). Mass spectrum: [M+1]515.3.

[0288] Example 4B

[0289]

[0290] Using intermediate 9 and N,N-dimethyl-β-alanine as raw materials, Example 4B was obtained under the same conditions as the experimental steps described in step 5 of Example 1A.

[0291] The NMR data are as follows: 1 H NMR (400MHz, CDCl3) δ7.77(d,J=7.2Hz,1H),4.41(dd,J=14.1,7.5Hz,1H),3.95(dd,J=10.9,2.3Hz,1H),3.78–3.65(m,1H),3.30(d,J=11.0H z,1H),2.53(t,J=6.2Hz,2H),2.32(t,J=6.2Hz,2H),2.27(s,6H),1.23-2.07(m,27H),1.08(d,J=7.1Hz,3H),1.02–0.93(m,6H),0.76(s,3H). Mass spectrum: [M+1]515.3.

[0292] Example 5

[0293]

[0294] Using intermediate 3 and 3-oxo-3-(4-methylpiperazin-1-yl)propionic acid as raw materials, Example 5 was obtained under the same conditions as the experimental steps described in step 4 of Example 1.

[0295] The NMR data are as follows: 1HNMR (CDCl3, 400MHz): δ 0.76 (s, 3H), 0.80–2.20 (m, 36H), 2.40 (s, 3H), 2.55–2.70 (m, 4H), 3.25–4.20 (m, 9H), 4.45–4.55 (m, 1H); Mass spectrometry: [M+1] 584.3.

[0296] Example 6

[0297]

[0298] Using intermediate 3 and 3-oxo-3-(1-methylpiperazine-4-amino)propionic acid as raw materials, Example 6 was obtained under the same conditions as the experimental steps described in step 4 of Example 1.

[0299] The NMR data are as follows: 1 ¹H NMR (CDCl₃, 400 MHz): δ 0.76 (s, 3H), 0.80–2.20 (m, 40H), 2.75–3.70 (m, 12H), 3.25–3.70 (m, 2H), 3.84–4.40 (m, 3H), 7.60–7.80 (m, 5H); Mass spectrometry: [M+1] 598.3.

[0300] Example 7

[0301]

[0302] Using intermediate 3 and 3-oxo-3-(1-methylpiperidin-4-methylamino)propionic acid as raw materials, Example 7 was obtained under the same conditions as the experimental steps described in step 4 of Example 1.

[0303] The NMR data are as follows: 1 ¹H NMR (CDCl₃, 400 MHz): δ 0.76 (s, 3H), 0.80–2.30 (m, 41H), 2.75–3.00 (m, 4H), 3.25–3.70 (m, 11H), 3.84–4.40 (m, 2H), 7.60–7.70 (m, 1H); Mass spectrometry: [M+1] 612.3.

[0304] Example 8

[0305]

[0306] Using intermediate 3 and 3-oxo-3-(3-morpholinopropyl)aminopropionic acid as raw materials, Example 8 was obtained under the same conditions as the experimental steps described in step 4 of Example 1.

[0307] The NMR data were as follows: 1H NMR (CDCl3, 400MHz): δ 0.75 (s, 3H), 0.80–2.30 (m, 38H), 2.45–2.60 (m, 4H), 3.25–4.00 (m, 13H), 4.10–4.55 (m, 2H), 7.60–7.80 (m, 1H); Mass spectrometry: [M+1] 628.3.

[0308] Example 9

[0309]

[0310] Using intermediate 3 and 3-oxo-3-(1,4-bispiperidine-1-)propionic acid as raw materials, Example 9 was obtained under the same conditions as the experimental steps described in step 4 of Example 1.

[0311] NMR data: 1H NMR (CDCl3, 400MHz, ppm): δ 0.76 (s, 3H), 0.80–2.30 (m, 46H), 2.55–3.60 (m, 10H), 3.75–4.55 (m, 5H), 7.20–7.40 (m, 1H); Mass spectrometry: [M+1] 652.3.

[0312] Example 10

[0313]

[0314] Using intermediate 3 and 3-oxo-3-(piperazine-1-)propionic acid as raw materials, Example 10 was obtained under the same conditions as the experimental steps described in step four of Example 1.

[0315] The NMR data were as follows: 1H NMR (CDCl3, 400MHz): δ 0.77 (s, 3H), 0.80–2.20 (m, 36H), 2.85–4.00 (m, 13H), 4.40–4.55 (m, 1H), 7.40–7.40 (m, 1H); Mass spectrometry: [M+1] 570.3.

[0316] Example 11

[0317]

[0318] Using intermediate 3 and 3-(hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanoic acid as raw materials, Example 11 was obtained under the same conditions as the experimental steps described in step 4 of Example 1.

[0319] NMR data: 1H NMR (CDCl3, 400MHz): δ 0.76 (s, 3H), 0.80–2.20 (m, 38H), 2.95–4.00 (m, 13H), 4.40–4.55 (m, 1H), 7.20–7.50 (m, 2H); Mass spectrometry: [M+1] 596.4.

[0320] Example 12

[0321]

[0322] Using intermediate 3 and 3-oxo-3-(4-(2-(pyrrolidin-1-yl)ethyl)piperazin-1-yl)propanoic acid as raw materials, Example 12 was obtained under the same conditions as the experimental steps described in step four of Example 1.

[0323] NMR data: 1H NMR (CDCl3, 400MHz): δ 0.74 (s, 3H), 0.80–2.30 (m, 36H), 2.45–2.80 (m, 8H), 3.25–4.00 (m, 11H), 4.10–4.45 (m, 2H); Mass spectrometry: [M+1] 667.5.

[0324] Example 13

[0325]

[0326] The preparation process of Example 13 is as follows:

[0327] Step 1: Intermediate 10

[0328]

[0329] In a 500 mL round-bottom flask, intermediate 12 (5 g, 1 equivalent), mono-tert-butyl malonate (2 equivalents), EDC·HCl (2 equivalents), DMAP (0.1 equivalents), and Et3N (5 equivalents) were added and dissolved in 100 mL of dry DCM. The mixture was stirred at room temperature for 2 h, and the reaction was monitored by TLC until completion (PMA colorimetry). After the reaction was complete, the mixture was washed twice with saturated NH4Cl aqueous solution, dried, and purified by silica gel column chromatography to obtain 4.5 g of product.

[0330] Step 2: Intermediate 11

[0331]

[0332] Intermediate 10 (4.5 g, 1 equivalent) was added to a 500 mL round-bottom flask and dissolved in 100 mL of dry DCM. TFA (10 mL, 15 equivalent) was added dropwise at 0 °C. After 15 min, the mixture was allowed to rise to room temperature and reacted for 1–2 h. The reaction was monitored by TLC until completion (PMA colorimetric analysis). After the reaction was complete, the mixture was evaporated to dryness, reconstituted with DCM, washed twice with saturated NaHCO3 aqueous solution, dried, and purified by silica gel column chromatography to obtain 4 g of the white solid product, which is intermediate 11.

[0333] Step 3: Example 13

[0334]

[0335] In a 25 mL round-bottom flask, intermediate 11 (50 mg, 1 equivalent), intermediate 12 (2 equivalents), HATU (2 equivalents), and Et3N (5 equivalents) were added and dissolved in 3 mL of dry DCM. The mixture was stirred at room temperature for 1 h, and the reaction was monitored by TLC until completion (PMA colorimetric assay). After the reaction was complete, the mixture was washed twice with saturated NH4Cl aqueous solution, dried, and subjected to thin-layer chromatography (dichloromethane:methanol = 10:1) to obtain 8 mg of a white solid.

[0336] The NMR data are as follows: 1H NMR (CDCl3, 400MHz, ppm): δ0.76 (s, 3H), δ0.80-2.30 (m, 36H), δ2.55-3.00 (m, 9H), δ3.15-4.50 (m, 14H), δ5.25 (br s, 1H); Mass spectrometry: [M+1]641.5.

[0337] Example 13A

[0338] Step 1 intermediate 13

[0339]

[0340] Raw material A (460 g, 1.10 mol, 1.0 eq) was dissolved in pyridine (6.9 L) and cooled to 0 ± 5 °C. TsCl (210.5 g, 1.10 mol, 1.0 eq) was dissolved in pyridine (600 mL) and slowly added to the reaction solution under N2 protection. The reaction solution was naturally heated to room temperature (23–28 °C). After stirring for 4 h, 105 g of pyridine (200 mL) solution of TsCl (0.55 mol, 0.5 eq) was added to the reaction solution under N2 protection at room temperature. After the addition was complete, the reaction was allowed to proceed overnight at room temperature (23–28 °C). The system turned wine red and was stirred at room temperature (23–28 °C) for 48 h. After the reaction was completed, the reaction solution was slowly dripped into water (65L) at 0-10℃. A large amount of solid precipitated out. The mixture was filtered, and the filter cake was washed with 500mL of PE:EA (50:1) mixed solvent. The obtained solid was dried under vacuum (40-50℃ water bath) for 8 hours to obtain 451g of solid, which is intermediate 13.

[0341] Second step intermediate 14

[0342]

[0343] To a 500 mL flask, add intermediate 13 (25 g, 43.8 mmol), potassium acetate (8.60 g, 87.6 mmol), and 18-crown-6 (23.15 g, 87.6 mmol), along with 300 mL of dimethyl sulfoxide solvent. Heat the reaction mixture to 55 °C and maintain this temperature for 16 hours. Monitor the reaction by TLC (petroleum ether:ethyl acetate = 15:1) until complete. Then, pour the reaction mixture into 1 L of ice water and stir for 30 minutes. Filter and wash the filter cake with water to give 7 g of white solid.

[0344] Third step intermediate 5

[0345]

[0346] To a 250 mL flask, add intermediate 14 (2.0 g, 4.36 mmol), 60 mL tetrahydrofuran, 60 mL methanol, 30 mL water, and 5.5 mL 4N LiOH aqueous solution. Heat the reaction mixture to 60 °C and maintain this temperature for 2 hours. Monitor the reaction using TLC; the developing solution was petroleum ether:ethyl acetate = 7:1. After the reaction was complete, remove most of the organic solvent by rotary evaporation and add 50 mL of water. Filter and wash the filter cake with water to give 1.7 g of a white solid.

[0347] Step 4 Intermediate 15

[0348]

[0349] To a 100 mL reaction flask, add intermediate 5 (5.0 g, 12.0 mmol), TsCl (11.4 g, 60.0 mmol), and DMAP (73 mg, 0.6 mmol), and dissolve in 50 mL of pyridine. Heat the reaction mixture to 50 °C and maintain this temperature for 16 hours. Monitor the reaction by TLC with a PE:EA ratio of 7:1 as the developing solvent. After the reaction is complete, pour the reaction mixture into 150 mL of water and stir for 30 minutes. Filter the reaction mixture and wash the filter cake with water to obtain 5.5 g of a white solid.

[0350] Fifth step intermediate 16

[0351]

[0352] TMSN3 (0.12 g, 1.1 eq, 2 eq) and DBU (0.32 g, 0.21 mmol, 4 eq), followed by N2, were added dropwise to a DMF suspension (6 mL) containing 15 (0.3 g, 0.52 mmol, 1 eq) at 30–35 °C. The resulting reaction solution was heated to 80–90 °C and reacted for 16–20 hours. After TLC detection showed that 5 had essentially disappeared (PE:EA = 10:1), the reaction solution was cooled to 30–35 °C. The reaction solution was then transferred to 20 mL of water at 0–10 °C and stirred for 5–10 minutes, followed by filtration. The filter cake was purified by silica gel column chromatography, eluting PE:EA, to give 0.13 g of solid, intermediate 16, with a yield of 56%.

[0353] Step 6 Intermediate 7

[0354]

[0355] Under nitrogen protection, intermediate 16 (12.0 g, 27.2 mmol) and 100 mL of dichloromethane solvent were added to a flask. 4.8 g of Pd / C and 200 mL of ethanol were also added. After purging with hydrogen (50 psi), the reaction mixture was heated to 50°C and maintained at this temperature for 16 hours. The reaction was monitored by TLC with petroleum ether:ethyl acetate as the developing solvent (20:1). The reaction mixture was filtered, and the filter cake was passed through a column (dichloromethane:methanol = 20:1) to give 11 g of a white solid, intermediate 7.

[0356] Step 7 Intermediate 17

[0357]

[0358] Using intermediate 7 as raw material, under the same conditions described for intermediate 10, a total of 40.4g of intermediate 17 was obtained.

[0359] Step 8 Intermediate 18

[0360]

[0361] Using intermediate 17 as raw material, and under the same conditions as described for intermediate 11, intermediate 18 (35g) was obtained.

[0362] Step 9, Example 13A

[0363]

[0364] Intermediate 18 (28 g, 1.0 eq) was dissolved in DCM (300 mL), and intermediate 12 (13.1 g, 1.5 eq), T3P (35.3 g, 2.0 eq) and NMM (11.2 g, 2.0 eq) were added. The mixture was reacted at room temperature for 16 h. TLC detection (DCM / MeOH (5% ammonia-methanol) = 20 / 1). After the reaction was complete, the reaction solution was washed with (250 mL) water, and the aqueous phase was extracted with DCM (100 mL x 2). The combined organic phases were dried with anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (DCM / MeOH (5% ammonia-methanol) = 45 / 1 to 15 / 1) to obtain approximately 17 g of crude product of Example 13A. This crude product was dissolved in approximately 120 mL of dichloromethane, and then acetonitrile (200 mL) was added. The solution was concentrated under reduced pressure to approximately 100 mL and stirred in an ice bath for 2 hours, resulting in the precipitation of a large amount of white solid. The reactants were filtered, the filter cake was dried, and then placed in pure water (150 mL) at room temperature and stirred overnight. After filtration and drying, Example 13A (12.2 g) was obtained.

[0365] The NMR data are as follows: ¹H NMR (400 MHz, Chloroform-d) δ 8.01 (d, J = 7.7 Hz, ¹H), 4.41 (td, J = 7.8, 5.9 Hz, ¹H), 4.18 (s, ¹H), 3.95 (dd, J = 11.0, 2.8 Hz, ¹H), 3.66 (d, J = 5.7 Hz, 2H), 3.57 (t, J = 5.1 Hz, 2H), 3.32–3.30 (m,1H),3.29(d,J=3.7Hz,2H),2.55(s,4H),2.51–2.44(m,4H),2.37(m,6H),2.10–1.66(m ,12H),1.66–1.45(m,7H),1.33–1.10(m,8H),1.08(m,3H),1.03–0.95(m,6H),0.75(s,3H). Mass spectrum: [M+1]641.5.

[0366] Example 13B

[0367]

[0368] A mixture of intermediate 13 (240 g, 0.42 mol, 1 eq) and 15-crown-5 (277.8 g, 1.2 mol, 3 eq) in DMSO (12 L) was stirred until completely dissolved. NaN3 (81.9 g, 1.26 mol, 3 eq) was added under N2 protection at 25–35 °C. The resulting mixture was heated to 60–70 °C and reacted for 2.5–3 hours under N2 protection. A sample was taken and analyzed by TLC (PE:EA = 9:1). The starting material disappeared. After the reaction solution temperature was lowered to 20–30 °C, water was added, and a solid precipitated. The solid was filtered, and the filter cake was washed with 2 L of water and dried to obtain 177 g of white solid intermediate 19.

[0369] Second step intermediate 23

[0370]

[0371] Intermediate 19 (650g) was dissolved in 10L of DCM and 10L of MeOH, and the solution was transferred to an autoclave. 104g of 10% wet Pd / C was suspended in 200mL of MeOH and transferred to the autoclave. The autoclave was purged with N2 four times at 28–35℃, followed by purging with H2 four times at the same temperature. Finally, the H2 pressure was maintained at 2.5–3MPa, and the mixture was stirred at 28–35℃ for 48 hours. TLC (PE:EA = 20:1) was performed to detect the disappearance of the starting material. After the reaction was complete, a layer of diatomaceous earth was placed on a vacuum filtration funnel, and the mixture was filtered under reduced pressure to remove Pd / C from the reaction solution. The filter cake was purified by column chromatography to obtain 460g of a white solid intermediate 9.

[0372] Step 3 Intermediate 20

[0373]

[0374] Using intermediate 9 as raw material, and following the same experimental steps described for intermediate 10, a total of 40.4 g of intermediate 20 was obtained.

[0375] Step 4 Intermediate 21

[0376]

[0377] Using intermediate 20 as raw material, crude intermediate 21 (35g) was obtained by following the same experimental steps described for intermediate 11.

[0378] Fifth step, Example 13B

[0379]

[0380] Intermediate 21 (28 g, 1.0 eq) was dissolved in DCM (300 mL), and intermediate 12 (13.1 g, 1.5 eq), T3P (35.3 g, 2.0 eq), and NMM (11.2 g, 2.0 eq) were added. The reaction was carried out at room temperature for 16 h. The reaction was detected by TLC (DCM / MeOH (5% ammonia methanol) = 20 / 1). After the reaction was completed, 250 mL of water was added, and the aqueous phase was extracted with DCM (100 mL x 2). The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (DCM / MeOH (5% ammonia methanol) = 45 / 1 to 15 / 1) to obtain Example 13B (12.2 g).

[0381] NMR data: 1H NMR (400MHz, Chloroform-d) δ 7.39 (d, J = 8.2Hz, 1H), 4.42 (td, J = 7.6, 6.0Hz, 1H), 3.96 (dd, J = 11.0, 2.8Hz, 1H), 3.87–3.70 (m, 1H), 3.65 (t, J = 5.2Hz, 2H), 3.56 (q, J = 5.2Hz, 2H), 3.31 (d, J = 11.0Hz, 1H), 3.28 (s, 2H),2.59–2.36(m,8H),2.28(s,6H),2.13–1.75(m,11H),1.75–1.49(m,7H),1.49–1.30(m,7H),1.30–1. 11(m,7H),1.08(d,J=7.1Hz,4H),1.04(d,J=3.3Hz,1H),1.00(d,J=6.5Hz,3H),0.94(s,3H),0.75(s,3H). Mass spectrum: [M+1]641.5.

[0382] Example 14

[0383]

[0384] Step 1: Example 14

[0385]

[0386] In a 25 mL round-bottom flask, intermediate 11 (50 mg, 1 eq), aminoethanol (2 eq), HATU (2 eq), and Et3N (5 eq) were added and dissolved in 3 mL of dry DCM. The mixture was stirred at room temperature for 1 h, and the reaction was monitored by TLC until completion (PMA colorimetric assay). After the reaction was complete, the mixture was washed twice with saturated NH4Cl aqueous solution, dried, and subjected to thin-layer chromatography (dichloromethane:methanol = 10:1) to obtain 38 mg of a white solid as described in Example 14.

[0387] The NMR data are as follows: ¹H NMR (CDCl₃, 400 MHz, ppm): 0.75 (s, 3H), 0.80–2.10 (m, 36H), 3.10–3.70 (m, 7H), 3.80–4.15 (m, 2H), 4.40–4.55 (m, 1H), 7.10–7.40 (m, 2H); Mass spectrometry: [M+1] 545.5 Example 14A

[0388]

[0389]

[0390] Using intermediate 18, the same synthesis method as in Example 14 can be used to obtain the white solid Example 14A.

[0391] The NMR data are as follows: 1H NMR (400MHz, Chloroform-d) δ 7.21 (s, 1H), 7.07 (d, J = 7.7Hz, 1H), 4.41 (q, J = 7.5Hz, 1H), 4.18 (s, 1H), 3.95 (dd, J = 11.0, 2.8Hz, 1H), 3.74 (q, J = 5.1Hz, 2H), 3.45 (td, J = 5. 6, 4.5 Hz, 2H), 3.30 (d, J = 11.0 Hz, 1H), 3.18 (s, 2H), 2.54 (t, J = 5.4 Hz, 1H), 2.09–1.60 (m, 11H), 1.53–1.11 (m, 14H), 1.08 (d, J = 7.1 Hz, 4H), 1.03–0.96 (m, 5H), 0.76 (s, 3H). Mass spectrometry: [M+1] 545.5

[0392] Example 14B

[0393]

[0394] Using intermediate 21, a white solid, Example 14B, can be obtained by the same synthesis method as in Example 14.

[0395] The NMR data are as follows: 1H NMR (400MHz, Chloroform-d) δ 7.42 (s, 1H), 6.67 (s, 1H), 4.41 (td, J = 7.8, 6.2Hz, 1H), 3.96 (dd, J = 11.0, 2.8Hz, 1H), 3.84–3.66 (m, 3H), 3.44 (td, J = 5.6, 4.4Hz, 2H), 3.35–3.22 (m, 1H). ),3.16(s,2H),2.81(s,1H),2.00(m,2H),1.92-1.75(m,5H),1.75-1.50(m,9H),1.50-1.3 1(m,7H),1.31-1.13(m,6H),1.08(m,3H),1.00(d,J=6.6Hz,3H),0.95(s,3H),0.75(s,3H).

[0396] Mass spectrometry: [M+1] 545.5

[0397] Example 15

[0398]

[0399] Using isoamyl saponin as raw material, Example 15 was obtained by the same method as in Example 14.

[0400] NMR data: 1H NMR (400MHz, Chloroform-d) δ 7.48 (s, 1H), 7.40 (d, J = 7.0Hz, 1H), 6.82 (s, 1H), 4.47–4.33 (m, 1H), 4.17 (s, 1H), 3.74 (m, 2H), 3.52–3.41 (m, 3H), 3.38 (m, 1H), 3.19 (d, J = 14.7Hz, 2H), 3.07 (q, J = 7.4Hz, 2H), 2.08–1.94 (m ,2H), 1.94-1.81(m,2H), 1.81-1.66(m,4H), 1.60(dd,J=16.2,11.9Hz,5H), 1.56-1.41(m,5H), 1.31-1.10(m,6H), 1.10-1.02(m,2H), 1.00(s,1H), 0.99-0.92(m,4H), 0.79(dd,J=6.3,1.5Hz,3H), 0.75(d,J=1.8Hz,3H). Mass spectrometry: [M+1]545.5 Example 15A

[0401]

[0402] Using isoamyl saponin as raw material, Example 15A was obtained by the same method as in Example 14A.

[0403] NMR data: 1H NMR (400MHz, Chloroform-d) δ 7.40 (s, 1H), 7.30 (d, J = 7.6Hz, 1H), 4.40 (td, J = 7.7, 5.9Hz, 1H), 4.17 (s, 1H), 3.73 (d, J = 4.8Hz, 2H), 3.41 (m, 4H), 3.20 (s, 2H), 2.92 (s, 1H), 1.43 (m, 33H), 0.78 (m, 6H). Mass spectrometry: [M+1] 545.5

[0404] Example 15B

[0405]

[0406] Using isoamyl saponin as raw material, Example 15B was obtained by the same method as in Example 14A.

[0407] NMR data: 1H NMR (400MHz, Chloroform-d) δ 7.51 (s, 1H), 6.83 (d, J = 7.9Hz, 1H), 4.41 (q, J = 7.4Hz, 1H), 3.74 (m, 3H), 3.42 (m, 4H), 3.17 (s, 2H), 1.38 (m, 34H), 0.78 (m, 6H). Mass spectrometry: [M+1] 545.5

[0408] Example 16A

[0409]

[0410] Using isoamyl saponin as raw material, Example 16A was obtained by the same method as in Example 13A.

[0411] The NMR data are as follows: ¹H NMR (400MHz, Chloroform-d) δ 8.02 (d, J = 7.7Hz, ¹H), 4.46–4.33 (m, ¹H), 4.18 (d, J = 7.3Hz, ¹H), 3.66 (dq, J = 5.7, 3.1, 2.2Hz, 2H), 3.57 (q, J = 5.2, 4.6Hz, 2H), 3.47 (ddd, J = 10.9, 4.4, 2.0Hz, 1H), 3.37 (t, J = 10.9Hz, 1H), 3.3 0(d,J=3.4Hz,2H),2.52(d,J=2.9Hz,4H),2.48(t,J=6.1Hz,4H),2.33(d,J=6.0Hz,7H),2.07-1.28(m,29H),1 .23-1.02(m,10H),1.00(s,3H),0.96(d,J=6.9Hz,4H),0.92-0.81(m,5H),0.79(d,J=6.3Hz,3H),0.76(s,3H). Mass spectrum: [M+1]641.5.

[0412] Example 16B

[0413]

[0414] Using isoamyl saponin as raw material, Example 16B was obtained through the same preparation steps and conditions as in Example 13B.

[0415] NMR data were as follows: ¹H NMR (400 MHz, Chloroform-d) δ 7.40 (d, J = 8.1 Hz, ¹H), 4.49–4.29 (m, ¹H), 3.88–3.70 (m, ¹H), 3.65 (t, J = 5.1 Hz, 2H), 3.60–3.53 (m, 2H), 3.53–3.45 (m, 1H), 3.38 (t, J = 10.9 Hz, 1H), 3.28 (s, 2H), 2.55–2.39 (m, 8H), 2.26 (s, 6H), 2.02–1.01 (m, 38H), 1.01–0.90 (m, 6H), 0.79 (d, J = 6.3 Hz, 3H), 0.75 (s, 3H). Mass spectrometry: [M+1] 641.5.

[0416] Example 17A

[0417]

[0418] Using isoamycin as a raw material, intermediate 22 was obtained through the same preparation steps and conditions as intermediate 7; using intermediate 22 as a raw material, Example 17A was obtained through the same preparation method and conditions as described in step five of Example 1A. Mass spectrometry: [M+1] 570.

[0419] Example 17B

[0420]

[0421] Using isoamycin as a raw material, intermediate 23 was obtained through the same preparation steps and conditions as intermediate 9; using intermediate 23 as a raw material, Example 17B was obtained through the same preparation method and conditions as described in Example 1B.

[0422] Mass spectrometry: [M+1]570.

[0423] Example 18A

[0424]

[0425] Using isopropionate as a raw material, Example 18A was obtained by following the same preparation steps and conditions as described in Example 4A.

[0426] The NMR data are as follows: 1H NMR (400MHz, Chloroform-d) δ 9.14 (s, 1H), 4.45–4.34 (m, 1H), 4.19 (d, J = 8.3 Hz, 1H), 3.52–3.42 (m, 2H), 3.37 (t, J = 10.9 Hz, 1H), 2.56 (t, J = 5.9 Hz, 2H), 2.36 (dd, J = 6.5, 5.2H). z, 2H), 2.31 (s, 6H), 2.09–1.66 (m, 10H), 1.66–1.48 (m, 8H), 1.48–1.35 (m, 7H), 1.34–1.00 (m, 14H), 0.99–0.93 (m, 7H), 0.92–0.82 (m, 4H), 0.79 (d, J = 6.3 Hz, 3H), 0.76 (s, 3H). Mass spectrometry: [M+1]515.

[0427] Example 18B

[0428]

[0429] Using isopropionate as raw material, Example 18B was obtained by following the same preparation steps and conditions as described in Example 4B.

[0430] The NMR data are as follows: 1H NMR (400MHz, Chloroform-d) δ 6.46 (s, 1H), 4.46–4.34 (m, 1H), 3.81–3.60 (m, 1H), 3.47 (d, J = 4.6 Hz, 1H), 3.38 (t, J = 10.9 Hz, 1H), 3.26 (t, J = 6.6 Hz, 2H), 2.80 (t, J = 6.6 Hz, 2H). ), 2.72(s, 6H), 2.05-1.53(m, 16H), 1.46(d, J = 4.3Hz, 2H), 1.37-1.33(m, 2H), 1.19-1.00(m, 9H), 0.99-0.91(m, 7H), 0.91-0.81(m, 14H), 0.79(d, J = 6.3Hz, 3H), 0.75(s, 3H). Mass spectrometry: [M+1]515.

[0431] Example 19A

[0432]

[0433]

[0434] Using hecosaponin as a raw material, intermediate 24 was obtained through the same preparation steps and conditions as intermediate 7; using intermediate 24 as a raw material, Example 19A was obtained through the same preparation method and conditions as described in step five of Example 1A.

[0435] The NMR data are as follows: ¹H NMR (400MHz, Chloroform-d) δ 4.33 (t, J = 7.4Hz, ¹H), 3.79–3.61 (m, 3H), 3.50 (s, 2H), 3.35 (t, J = 10.9Hz, 2H), 3.10 (q, J = 7.4Hz, 2H), 2.99 (s, 7H), 2.70 (s, 1H), 2.62–2.45 (m, 5H), 2.38 (t, J = 13.7Hz, 2H), 2.22 (dd, J = 14.3, 5.0Hz, 2H), 2. 11(q,J=6.5,6.0Hz,2H),1.90(d,J=9.0Hz,3H),1.75(dt,J=12.9,6.5Hz,5H),1.69-1.50(m,11H),1.44(dd,J=11.3, 7.9Hz, 6H), 1.40-1.09 (m, 10H), 1.09-1.01 (m, 5H), 0.97 (dd, J = 12.9, 7.9Hz, 2H), 0.89 (s, 4H), 0.79 (d, J = 6.3Hz, 3H). Mass spectrum: [M+1]529.4.

[0436] Example 20A

[0437]

[0438] Using hecosaponin as raw material, Example 20A was obtained by following the same preparation steps and conditions as described in Example 4A.

[0439] The NMR data are as follows: ¹H NMR (400MHz, Chloroform-d) δ 6.99 (t, J = 6.4Hz, ¹H), 4.33 (dd, J = 8.5, 5.8Hz, ¹H), 3.81–3.57 (m, ¹H), 3.57–3.43 (m, ¹H), 3.35 (t, J = 11.0Hz, ¹H), 3.15 (t, J = 6.5Hz, 2H), 2.71 (t, J = 6.5Hz, 2H), 2.66 (s, 6H), 2.51 (dd, J = 8.8, 6.7Hz, ¹H), 2.3 8(t,J=13.7Hz,1H),2.21(dd,J=14.3,5.0Hz,1H),2.11(t,J=7.1Hz,1H),1.89(dd,J=11.1,7.3Hz,1H),1.83-1.71(m, 3H),1.71-1.52(m,6H),1.52-1.38(m,3H),1.38-1.09(m,7H),1.09-0.99(m,6H),0.89(s,3H),0.79(d,J=6.3Hz,3H). Mass spectrum: [M+1]529.4.

[0440] Example 21

[0441]

[0442] Using 4-hydroxyphenylethylamine as a raw material, Example 21 was obtained by following the same preparation steps and conditions as described in Example 13.

[0443] NMR data were as follows: ¹H NMR (CDCl₃, 400 MHz, ppm): 0.76 (s, 3H), 0.80–2.10 (m, 36H), 2.65–2.80 (m, 2H), 3.15–3.60 (m, 5H), 3.70–4.55 (m, 4H), 6.70–7.40 (m, 5H); Mass spectrometry: [M+1] 621.4

[0444] Example 22

[0445]

[0446] Using 3,4-dihydroxyphenylethylamine as a raw material, Example 22 was obtained by following the same preparation steps and conditions as described in Example 13.

[0447] The NMR data are as follows: ¹H NMR (CDCl₃, 400 MHz, ppm): 0.7 (s, 3H), 0.80–2.10 (m, 36H), 2.45–2.55 (m, 2H), 3.25–3.70 (m, 6H), 3.84–4.55 (m, 3H), 6.50–6.90 (m, 4H); Mass spectrometry: [M+1] 637.4

[0448] Example 23

[0449]

[0450] The specific preparation process is as follows:

[0451] Step 1: Intermediate 25

[0452]

[0453] Under argon protection, 0.2 g of morpholine, 0.4 g of 1-Boc-3-azacyclobutanone, 0.7 mL of acetic acid, and 0.3 g of sodium cyanoborohydride were dissolved in 20 mL of dichloromethane and reacted at room temperature for 2 h. The reaction was then detected. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 70%.

[0454] Step 2: Intermediate 26

[0455]

[0456] Under argon protection, 0.2 g of intermediate 25 was dissolved in 10 mL of dichloromethane, and 5 mL of dioxane hydrochloride solution was added. The mixture was reacted at room temperature for 2 h, and the reaction was monitored. The solvent was evaporated and the mixture was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 90%.

[0457] Step 3: Synthesis of Example 23

[0458]

[0459] Under argon protection, 0.1 g of intermediate 26, 0.43 g of intermediate 11, 0.40 g of HATU, and 0.22 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 60%.

[0460] NMR data are as follows: 1H NMR (400MHz, CD3OD) δ 4.5-4.65 (m, 2H), 4.26-4.45 (m, 2H), 4.05-4.25 (m, 3H), 3.85-3.99 (m, 2H), 3.72-3.85 (m, 1H), 3.60-3.70 (m, 1H), 3.40-3.60 (m, 2H), 3.06-3.28 (m, 3H), 2.71 (s, 4H), 1.32-2.10 (m, 27H), 1.08 (dd, 3H), 0.99 (m, 6H), 0.75 (s, 3H). Mass spectrometry: [M+1] 626.5.

[0461] Example 24

[0462]

[0463] The specific preparation process is as follows:

[0464]

[0465] Under argon protection, 0.2 g of intermediate 11, 0.08 g of 1-methyl-4-piperidin-4-ylpiperazine, 0.227 g of HATU, and 0.101 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and vanillin staining. The yield was 45%.

[0466] NMR data: 1H NMR (400MHz, CDCl3) δ 8.13 (t, 0.5H), 7.44 (dd, 0.5H), 4.58 (d, 1H), 4.41 (p, 1H), 3.96 (m, 2H), 3.29 (d, 3H), 3.06 (dd, 1H), 2.43 (m, 15H), 1.37 (m, 39H), 0.75 (d, 3H). Mass spectrometry: [M+1] 667.5.

[0467] Example 24A, Example 24B

[0468]

[0469] Using intermediate 18, Example 24A can be obtained by the same synthesis method as in Example 24. Using intermediate 21, Example 24B can be obtained by the same method.

[0470] The NMR data for Example 24A are as follows: 1H NMR (400MHz, CDCl3) δ 8.12 (t, 1H), 4.48 (m, 2H), 4.00 (m, 3H), 3.28 (m, 5H), 2.43 (m, 13H), 1.82 (m, 15H), 1.12 (m, 27H). Mass spectrometry: [M+1] 667.5.

[0471] The NMR data for Example 24B are as follows: 1H NMR (400MHz, CDCl3) δ 7.42 (dd, 1H), 4.50 (m, 2H), 3.94 (m, 2H), 3.31 (d, 3H), 3.06 (m, 2H), 2.56 (m, 11H), 2.31 (s, 3H), 1.40 (m, 42H). Mass spectrometry: [M+1] 667.5.

[0472] Example 25

[0473]

[0474] The specific preparation process is as follows:

[0475]

[0476] The preparation method is the same as in Example 24.

[0477] NMR data: 1H NMR (400MHz, CDCl3) δ 7.75 (dt, 1H), 4.46 (m, 3H), 3.94 (m, 3H), 3.39 (m, 6H), 3.06 (m, 2H), 2.65 (m, 14H), 1.28 (m, 40H). Mass spectrometry: [M+1] 681.6.

[0478] Example 25A, Example 25B

[0479]

[0480] Example 25A can be obtained using intermediate 18 and 1-ethyl-4-(piperidin-4-methyl)piperazine, following the same synthetic method as in Example 24. Example 25B can be obtained using intermediate 21 and 1-ethyl-4-(piperidin-4-methyl)piperazine, following the same method.

[0481] The NMR data for Example 25A are as follows: 1H NMR (400MHz, CDCl3) δ 8.15 (t, 1H), 4.61 (d, 1H), 4.41 (m, 1H), 4.19 (s, 1H), 3.96 (m, 2H), 3.31 (d, 3H), 3.06 (t, 1H), 2.51 (m, 10H), 1.34 (m, 48H). Mass spectrometry: [M+1] 681.6.

[0482] The NMR data for Example 25B are as follows: 1H NMR (400MHz, CDCl3) δ 7.38 (d, 1H), 4.42 (q, 1H), 3.96 (dd, 1H), 3.62 (m, 5H), 3.31 (d, 3H), 2.90 (m, 3H), 2.54 (m, 4H), 2.37 (s, 3H), 1.38 (m, 45H). Mass spectrometry: [M+1] 681.6.

[0483] Example 26

[0484]

[0485] The specific preparation process is as follows:

[0486]

[0487] The preparation method is the same as in Example 24.

[0488] NMR data: 1H NMR (400MHz, CDCl3) δ 7.74 (dd, 1H), 4.41 (p, 1H), 3.96 (dt, 1H), 3.59 (m, 6H), 3.29 (d, 2H), 2.91 (m, 2H), 2.54 (q, 4H), 2.26 (s, 4H), 1.41 (m, 45H). Mass spectrometry: [M+1] 667.6.

[0489] Example 26A, Example 26B

[0490]

[0491] Example 26A can be obtained using intermediate 18 and 1-(1-methyl-4-piperidine)piperazine, following the same synthetic method as in Example 24. Example 26B can be obtained using intermediate 21 and 1-(1-methyl-4-piperidine)piperazine, following the same method.

[0492] The NMR data for Example 26A are as follows: 1H NMR (400MHz, CDCl3) δ 8.09 (d, 1H), 4.40 (q, 1H), 4.16 (d, 1H), 3.95 (dd, 1H), 3.64 (t, 2H), 3.49 (s, 5H), 3.29 (s, 2H), 2.91 (d, 2H), 2.54 (q, 4H), 2.27 (s, 3H), 1.42 (m, 44H). Mass spectrometry: [M+1] 667.6.

[0493] Magnetic data for Example 26B are as follows: ¹H NMR (400MHz, CDCl₃) δ 7.38 (d, ¹H), 4.42 (q, ¹H), 3.96 (dd, ¹H), 3.62 (m, 5H), 3.31 (d, 3H), 2.90 (m, 3H), 2.54 (m, 4H), 2.37 (s, 3H), 1.38 (m, 45H). Mass spectrometry: [M+1] 667.6.

[0494] Example 27

[0495]

[0496] The specific preparation process is as follows:

[0497] Step 1: Intermediate 27

[0498]

[0499] 0.5 g of tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylic acid and 0.714 g of di-tert-butyl dicarbonate were dissolved in 20 mL of dry DCM and reacted at room temperature for 12 h. The reaction was then monitored. After the reaction was complete, the solvent was evaporated, and the mixture was extracted with water / dichloromethane. The mixture was purified by silica gel column chromatography (DCM:MeOH = 30:1) and detected by TLC (DCM:MeOH = 10:1), with vanillin as the color indicator. The yield was 65%.

[0500] Step 2: Intermediate 28

[0501]

[0502] 15 mL of THF was added to 0.2 g of intermediate 27 and 0.07 g of LiAlH4, and the mixture was heated to reflux for 3 h. The reaction was then monitored. After the reaction was complete, the mixture was cooled to room temperature, and the solvent was evaporated to obtain the product. This product was then directly added to the next reaction, and vanillin was used for colorimetric analysis. The yield was 56%.

[0503] Step 3: Synthesis of Example 27

[0504]

[0505] Under argon protection, 0.037 g of intermediate 28, 0.12 g of intermediate 11, 0.136 g of HATU, and 0.061 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and vanillin staining. The yield was 55%.

[0506] NMR data are as follows: 1H NMR (400MHz, CDCl3) δ 7.97 (m, 1H), 4.11 (m, 3H), 3.48 (dt, 2H), 3.30 (m, 3H), 3.02 (d, 3H), 2.44 (m, 13H), 1.23 (m, 40H). Mass spectrometry: [M+1] 641.6.

[0507] Example 27A, Example 27B

[0508]

[0509] Its synthesis is the same as in Example 27, as follows:

[0510] Example 27A can be obtained by using intermediate 18 and N-methyl-2-(1-methylpiperidin-4-yl)ethane-1-amine in the same manner as in Example 27. Example 27B can be obtained by using intermediate 21 and N-methyl-2-(1-methylpiperidin-4-yl)ethane-1-amine in the same manner.

[0511] The NMR data for Example 27A are as follows: 1H NMR (400MHz, CDCl3) δ 8.45 (dd, 1H), 4.41-4.45 (m, 1H), 3.94-3.98 (m, 1H), 3.75-3.78 (m, 1H), 3.51 (t, 1H), 3.45 (t, 1H), 3.28-3.32 (m, 3H), 3.10 (m, 3H), 2.51 (m, 10H), 2.26 (m, 3H), 1.32-2.10 (m, 27H), 1.08 (m, 3H), 0.99 (m, 6H), 0.75 (s, 3H). Mass spectrometry: [M+1] 641.6.

[0512] The NMR data for Example 27B are as follows: 1H NMR (400MHz, CDCl3) δ 8.7.78 (dd, 1H), 4.41-4.45 (m, 1H), 3.94-3.98 (m, 1H), 3.75-3.78 (m, 1H), 3.51 (t, 1H), 3.45 (t, 1H), 3.28-3.32 (m, 3H), 3.10 (m, 3H), 2.51 (m, 10H), 2.26 (m, 3H), 1.32-2.10 (m, 27H), 1.08 (m, 3H), 0.99 (m, 6H), 0.75 (s, 3H). Mass spectrometry: [M+1] 641.6.

[0513] Example 28

[0514]

[0515] The specific preparation process is as follows:

[0516]

[0517] Under argon protection, 0.1 g piperazine-1-carboxylic acid methyl ester, 0.42 g intermediate 11, 0.40 g HATU, and 0.21 g TEA were dissolved in 10 mL dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 65%.

[0518] NMR data were as follows: 1H NMR (CDCl3-d6, 400MHz): 7.12 (d, 1H), 4.35-4.46 (m, 1H), 3.90-4.01 (m, 1H), 3.70-3.85 (m, 1H), 3.73 (s, 3H), 3.60-3.68 (m, 2H), 3.45-3.58 (m, 6H), 3.28-3.35 (m, 1H), 3.29 (s, 2H), 1.58-2.10 (m, 11H), 1.15-1.50 (m, 16H), 1.08 (d, 3H), 0.99 (d, 3H), 0.95 (s, 3H), 0.75 (s, 3H). Mass spectrometry: [M+1] 628.5.

[0519] Example 29

[0520]

[0521] The specific preparation process is as follows:

[0522] Step 1: Intermediate 29

[0523]

[0524] Under argon protection, 0.1 g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.05 g of bromoethane, and 0.08 g of TEA were dissolved in 30 mL of dichloromethane and reacted at room temperature for 12 h. The reaction was then monitored. The solvent was evaporated and the solution was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 80%.

[0525] Step 2: Intermediate 30

[0526]

[0527] Under argon protection, 0.1 g of intermediate 29 was dissolved in 10 mL of dichloromethane, and 5 mL of dioxane hydrochloride solution was added. The mixture was reacted at room temperature for 2 h, and the reaction was monitored. The solvent was evaporated and the mixture was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 90%.

[0528] Step 3: Synthesis of Example 29

[0529]

[0530] Under argon protection, 0.05 g of intermediate 30, 0.15 g of intermediate 11, 0.15 g of HATU, and 0.08 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 60%.

[0531] NMR data: 1H NMR (400MHz, CDCl3) δ 8.7.38 (dd, 1H), 4.41-4.45 (m, 1H), 3.94-3.98 (m, 1H), 3.75-3.78 (m, 1H), 3.65 (t, 2H), 3.56 (t, 2H), 3.28-3.32 (m, 3H), 3.15 (m, 2H), 2.51 (m, 6H), 2.30 (m, 1H), 2.10 (m, 6H), 1.32-2.10 (m, 27H), 1.08 (m, 3H), 0.99 (m, 9H), 0.75 (s, 3H). Mass spectrometry: [M+1] 681.5.

[0532] Example 29A, Example 29B

[0533]

[0534] The synthesis process is the same as in Example 29. Using intermediate 18 and 1-(1-ethylpiperidin-4-yl)piperazine, Example 29A can be obtained by the same synthesis method as in Example 29. Using intermediate 21 and 1-(1-ethylpiperidin-4-yl)piperazine, Example 29B can be obtained by the same method.

[0535] The NMR data for Example 29A are as follows: 1H NMR (400MHz, CDCl3) δ 8.8.05 (dd, 1H), 4.41-4.45 (m, 1H), 3.94-3.98 (m, 1H), 3.75-3.78 (m, 1H), 3.65 (t, 2H), 3.56 (t, 2H), 3.28-3.32 (m, 3H), 3.15 (m, 2H), 2.51 (m, 6H), 2.30 (m, 1H), 2.10 (m, 6H), 1.32-2.10 (m, 27H), 1.08 (m, 3H), 0.99 (m, 9H), 0.75 (s, 3H). Mass spectrometry: [M+1] 681.6.

[0536] The NMR data for Example 29B are as follows: ¹H NMR (400MHz, CDCl₃) δ 8.7.38 (dd, ¹H), 4.41-4.45 (m, ¹H), 3.94-3.98 (m, ¹H), 3.75-3.78 (m, ¹H), 3.65 (t, 2H), 3.56 (t, 2H), 3.28-3.32 (m, 3H), 3.15 (m, 2H), 2.51 (m, 6H), 2.30 (m, 1H), 2.10 (m, 6H), 1.32-2.10 (m, 27H), 1.08 (m, 3H), 0.99 (m, 9H), 0.75 (s, 3H). Mass spectrometry: [M+1] 681.5.

[0537] Example 30

[0538]

[0539] The specific preparation process is as follows:

[0540] Step 1: Intermediate 31

[0541]

[0542] Under argon protection, 0.4 g of intermediate 11, 0.258 g of 4-(piperidin-4-yl)piperazine-1-carboxylic acid tert-butyl ester, 0.455 g of HATU, and 0.202 g of TEA were dissolved in 15 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was evaporated, and the mixture was purified by silica gel column chromatography.

[0543] DCM:MeOH = 20:1, DCM:MeOH = 10:1 (for plate testing), vanillin color development, yield 75%.

[0544] Step 2: Intermediate 32

[0545]

[0546] Add 10 mL of MeOH to 0.2 g of intermediate 31, followed by 10 mL of 4 M HCl / dioxane solution, and react at room temperature for 3 h. Dry the solvent by rotary evaporation, and directly add to the next reaction step; vanillin is detected by color development. Yield: 80%.

[0547] Step 3: Synthesis of Example 30

[0548]

[0549] Under argon protection, 0.08 g of intermediate 32, 0.03 g of bromoacetonitrile, 0.034 g of potassium carbonate, and 0.02 g of potassium iodide were dissolved in 10 mL of acetonitrile. The mixture was heated to 50 °C and reacted for 12 h. The reaction was then monitored. The solution was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and vanillin staining. The yield was 65%.

[0550] NMR data: 1H NMR (400MHz, CDCl3) δ 7.37 (m, 1H), 4.58 (d, 1H), 4.42 (q, 1H), 3.98 (d, 1H), 3.77 (m, 1H), 3.50 (s, 2H), 3.30 (m, 2H), 3.07 (t, 1H), 2.62 (s, 12H), 1.12 (m, 43H). Mass spectrometry: [M+1] 693.6.

[0551] Example 31

[0552]

[0553] The specific preparation process is as follows:

[0554] Step 1: Intermediate 33

[0555]

[0556] 0.1 g of tert-butyl 4-(piperidin-4-yl)piperazine-1-carboxylic acid and 0.089 g of bromoacetonitrile were dissolved in 10 mL of dry MeCN. Then, 0.103 g of potassium carbonate and 0.062 g of potassium iodide were added. The mixture was heated to 50 °C and reacted for 12 h. The reaction was then monitored. After complete reaction, the mixture was extracted with water / dichloromethane and purified by silica gel column chromatography (DCM:MeOH = 30:1). The solution was then analyzed by TLC (DCM:MeOH = 10:1), and vanillin was detected. The yield was 55%.

[0557] Step 2: Intermediate 34

[0558]

[0559] Add 10 mL of MeOH to 0.2 g of intermediate 33, followed by 10 mL of 4 M HCl / dioxane solution, and react at room temperature for 3 h. Dry the solvent by rotary evaporation, and directly add to the next reaction step; vanillin is detected by color development. Yield: 80%.

[0560] Step 3: Synthesis of Example 31

[0561]

[0562] Under argon protection, 0.046 g of intermediate 34, 0.1 g of intermediate 11, 0.114 g of HATU, and 0.061 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and vanillin staining. The yield was 40%.

[0563] NMR data: 1H NMR (400MHz, CDCl3) δ 7.35 (m, 1H), 4.42 (m, 1H), 3.95 (m, 1H), 3.41 (m, 10H), 2.59 (m, 6H), 2.33 (ddd, 3H), 1.29 (m, 43H). Mass spectrometry: [M+1] 692.6.

[0564] Example 32

[0565]

[0566] The specific preparation process is as follows:

[0567] Step 1: Intermediate 35

[0568]

[0569] Under argon protection, 0.1 g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.06 g of bromoisobutane, and 0.08 g of TEA were dissolved in 30 mL of N,N-dimethylformamide. The mixture was heated to 50 °C and reacted for 12 h. The reaction was then monitored. The solvent was evaporated and the mixture was directly added to the next reaction step. Phosphomolybdic acid was used for colorimetric development. The yield was 70%.

[0570] Step 2: Intermediate 36

[0571]

[0572] Under argon protection, 0.1 g of intermediate 35 was dissolved in 10 mL of dichloromethane, and 5 mL of dioxane hydrochloride solution was added. The reaction was carried out at room temperature for 2 h, and the reaction was monitored. The solvent was evaporated and the mixture was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 90%.

[0573] Step 3: Synthesis of Example 36

[0574]

[0575] Under argon protection, 0.05 g of intermediate 36, 0.11 g of intermediate 11, 0.13 g of HATU, and 0.07 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 60%.

[0576] NMR data are as follows: 1H NMR (400MHz, CDCl3) δ 7.32 (dd, 1H), 4.41-4.45 (m, 1H), 3.94-3.98 (m, 1H), 3.75-3.78 (m, 1H), 3.65 (t, 2H), 3.56 (t, 2H), 3.28-3.32 (m, 3H), 2.5 (m, 5H), 2.3 (m, 2H), 1.32-2.10 (m, 27H), 1.19 (m, 9H), 1.08 (dd, 3H), 0.99 (m, 12H), 0.75 (s, 3H). Mass spectrometry: [M+1] 709.7.

[0577] Example 32A, Example 32B

[0578]

[0579] Its synthesis method is the same as 32

[0580] Example 32A can be obtained by using intermediate 18 and 1-(1-isobutylpiperidin-4-yl)piperazine in the same synthetic method as in Example 32. Example 32B can be obtained by using intermediate 21 and 1-(1-isobutylpiperidin-4-yl)piperazine in the same method.

[0581] The NMR data for Example 32A are as follows: 1H NMR (400MHz, CDCl3) δ 7.32 (dd, 1H), 4.41-4.45 (m, 1H), 3.94-3.98 (m, 1H), 3.75-3.77 (m, 1H), 3.65 (t, 2H), 3.56 (t, 2H), 3.28-3.32 (m, 3H), 2.52 (m, 5H), 2.32 (m, 2H), 1.32-2.09 (m, 27H), 1.19 (m, 9H), 1.09 (dd, 3H), 0.99 (m, 12H), 0.75 (s, 3H). Mass spectrometry: [M+1] 709.7.

[0582] The NMR data for Example 32B are as follows: 1H NMR (400MHz, CDCl3) δ 7.32 (dd, 1H), 4.41-4.45 (m, 1H), 3.94-3.98 (m, 1H), 3.75-3.78 (m, 1H), 3.65 (t, 2H), 3.56 (t, 2H), 3.28-3.32 (m, 3H), 2.51 (m, 5H), 2.31 (m, 2H), 1.32-2.10 (m, 27H), 1.19 (m, 9H), 1.08 (dd, 3H), 0.99 (m, 12H), 0.76 (s, 3H). Mass spectrometry: [M+1] 709.7.

[0583] Example 33

[0584]

[0585] The specific preparation process is as follows:

[0586] Step 1: Intermediate 37

[0587]

[0588] Under argon protection, 0.1 g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.22 g of intermediate 11, 0.21 g of HATU, and 0.11 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then detected. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 60%.

[0589] Step 2: Intermediate 38

[0590]

[0591] Under argon protection, 0.1 g of intermediate 37 was dissolved in 10 mL of dichloromethane, and 5 mL of dioxane hydrochloride solution was added. The reaction was carried out at room temperature for 2 h, and the reaction was monitored. The solvent was evaporated and the mixture was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 90%.

[0592] Step 3: Synthesis of Example 33

[0593]

[0594] Under argon protection, 0.1 g of intermediate 38, 0.03 g of bromoisobutane, and 0.03 g of TEA were dissolved in 15 mL of N,N-dimethylformamide. The mixture was heated to 50 °C and reacted for 12 h. The reaction was then monitored. The solvent was evaporated, and the mixture was purified by silica gel column chromatography (DCM:MeOH = 20:1). A TLC test was performed with a DCM:MeOH ratio of 10:1, and the mixture was developed with phosphomolybdic acid. The yield was 60%.

[0595] NMR data are as follows: 1H NMR (400MHz, CDCl3) δ 7.32 (dd, 1H), 4.52 (m, 1H), 4.48 (m, 1H), 3.94-3.98 (m, 1H), 3.75-3.78 (m, 1H), 3.26 (m, 3H), 3.15 (t, 1H), 2.25-2.75 (m, 10H), 2.2 (m, 2H), 1.32-2.10 (m, 27H), 1.19 (m, 6H), 1.08 (m, 9H), 0.75 (s, 3H). Mass spectrometry: [M+1] 709.6.

[0596] Example 33A, Example 33B

[0597]

[0598] The synthesis process is as follows: Example 33A can be obtained by using intermediate 18 and 1-bromo-2-methylpropane in the same manner as in Example 33. Example 33B can be obtained by using intermediate 21 and 1-bromo-2-methylpropane in the same manner.

[0599] NMR data are as follows: 1H NMR (400MHz, CDCl3) δ 7.32 (dd, 1H), 4.52 (m, 1H), 4.48 (m, 1H), 3.94-3.98 (m, 1H), 3.76-3.78 (m, 1H), 3.26 (m, 3H), 3.15 (t, 1H), 2.25-2.75 (m, 10H), 2.2 (m, 2H), 1.32-2.10 (m, 27H), 1.20 (m, 6H), 1.08 (m, 9H), 0.75 (s, 3H). Mass spectrometry: [M+1] 709.6.

[0600] The NMR data for Example 33B are as follows: ¹H NMR (400MHz, CDCl₃) δ 7.32 (dd, ¹H), 4.52 (m, ¹H), 4.48 (m, ¹H), 3.94-3.97 (m, ¹H), 3.75-3.78 (m, ¹H), 3.27 (m, 3H), 3.15 (t, ¹H), 2.25-2.75 (m, 10H), 2.2 (m, 2H), 1.32-2.10 (m, 27H), 1.19 (m, 6H), 1.09 (m, 9H), 0.75 (s, 3H). Mass spectrometry: [M+1] 709.5.

[0601] Example 34

[0602]

[0603] The specific preparation process is as follows:

[0604] Step 1: Intermediate 39

[0605]

[0606] Under argon protection, 0.1 g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.06 g of 2-bromopropane, and 0.08 g of TEA were dissolved in 30 mL of N,N-dimethylformamide. The mixture was heated to 50 °C and reacted for 12 h. The reaction was then monitored. The solvent was evaporated and the mixture was directly added to the next reaction step. Phosphomolybdic acid was used for colorimetric development. The yield was 70%.

[0607] Step 2: Intermediate 40

[0608]

[0609] Under argon protection, 0.1 g of intermediate 39 was dissolved in 10 mL of dichloromethane, and 5 mL of dioxane hydrochloride solution was added. The reaction was carried out at room temperature for 2 h, and the reaction was monitored. The solvent was evaporated and the mixture was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 90%.

[0610] Step 3: Synthesis of Example 34

[0611]

[0612] Under argon protection, 0.05 g of intermediate 40, 0.14 g of intermediate 11, 0.13 g of HATU, and 0.07 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 60%.

[0613] NMR data were as follows: 1H NMR (400MHz, CDCl3) δ 7.32 (dd, 1H), 4.41–4.45 (m, 1H), 3.94–3.98 (m, 1H), 3.75–3.78 (m, 1H), 3.51 (m, 6H), 3.28–3.32 (m, 3H), 2.55 (m, 8H), 1.32–2.10 (m, 27H), 1.19 (m, 14H), 1.08 (m, 6H), 0.99 (m, 3H), 0.75 (s, 3H). Mass spectrometry: [M+1] 695.7.

[0614] Example 34A, Example 34B

[0615]

[0616] The synthesis process is as follows: Example 34A can be obtained by using intermediate 18 and 1-(1-isopropylpiperidin-4-yl)piperazine in the same manner as in Example 34. Example 34B can be obtained by using intermediate 21 and 1-(1-isopropylpiperidin-4-yl)piperazine in the same manner.

[0617] The NMR data for Example 34A are as follows: 1H NMR (400MHz, CDCl3) δ 7.33 (dd, 1H), 4.41-4.45 (m, 1H), 3.94-3.98 (m, 1H), 3.75-3.79 (m, 1H), 3.51 (m, 6H), 3.28-3.32 (m, 3H), 2.55 (m, 8H), 1.32-2.11 (m, 27H), 1.19 (m, 14H), 1.08 (m, 6H), 0.99 (m, 3H), 0.75 (s, 3H). Mass spectrometry: [M+1] 695.7.

[0618] The NMR data for Example 34B are as follows: 1H NMR (400MHz, CDCl3) δ 7.32 (dd, 1H), 4.41-4.45 (m, 1H), 3.94-3.98 (m, 1H), 3.75-3.78 (m, 1H), 3.52 (m, 6H), 3.28-3.32 (m, 3H), 2.55 (m, 8H), 1.32-2.11 (m, 27H), 201.19 (m, 14H), 1.08 (m, 6H), 0.99 (m, 3H), 0.75 (s, 3H). Mass spectrometry: [M+1] 695.6.

[0619] Example 35

[0620]

[0621] The specific preparation process is as follows:

[0622]

[0623] Using intermediate 11 and 1-(pyridin-4-yl)piperazine as starting materials, Example 35 can be obtained by the same synthesis method as in Example 28.

[0624] NMR data were as follows: 1H NMR (400MHz, CDCl3) δ 8.25 (m, 2H), 7.15 (dd, 1H), 6.65 (m, 2H), 4.41–4.45 (m, 1H), 3.94–3.98 (m, 1H), 3.76 (m, 5H), 3.25–3.50 (m, 7H), 1.32–2.10 (m, 27H), 1.08 (m, 6H), 0.99 (m, 3H), 0.75 (s, 3H). Mass spectrometry: [M+1] 647.5.

[0625] Example 35A, Example 35B

[0626]

[0627] The synthesis process is as follows: Example 35A can be obtained by using intermediate 18 and 1-(pyridin-4-yl)piperazine in the same manner as in Example 28. Example 35B can be obtained by using intermediate 21 and 1-(pyridin-4-yl)piperazine in the same manner.

[0628] The NMR data for Example 35A are as follows: 1H NMR (400MHz, CDCl3) δ 8.24 (m, 2H), 7.14 (dd, 1H), 6.65 (m, 2H), 4.41-4.44 (m, 1H), 3.94-3.98 (m, 1H), 3.75 (m, 5H), 3.25-3.50 (m, 7H), 1.32-2.10 (m, 27H), 1.07 (m, 6H), 0.99 (m, 3H), 0.75 (s, 3H). Mass spectrometry: [M+1] 647.6.

[0629] The NMR data for Example 35B are as follows: 1H NMR (400MHz, CDCl3) δ 8.26 (m, 2H), 7.16 (dd, 1H), 6.66 (m, 2H), 4.41-4.45 (m, 1H), 3.94-3.99 (m, 1H), 3.77 (m, 5H), 3.25-3.50 (m, 7H), 1.32-2.11 (m, 27H), 1.08 (m, 6H), 0.99 (m, 3H), 0.75 (s, 3H). Mass spectrometry: [M+1] 647.5.

[0630] Example 36

[0631]

[0632] The specific preparation process is as follows:

[0633]

[0634] Using intermediate 11 and 1-(1-(2-fluoroethyl)piperidin-4-yl)piperazine as starting materials, Example 36 can be obtained by the same synthesis method as in Example 31.

[0635] NMR data were as follows: 1H NMR (400MHz, CDCl3) δ 8.09 (d, 1H), 4.62 (td, 1H), 4.50 (td, 1H), 4.41 (h, 1H), 4.19 (d, 1H), 3.95 (dt, 1H), 3.54 (m, 7H), 3.29 (d, 3H), 3.02 (dt, 3H), 2.61 (m, 8H), 2.30 (m, 1H), 1.34 (m, 39H). Mass spectrometry: [M+1] 699.6.

[0636] Example 37

[0637]

[0638] The specific preparation process is as follows:

[0639] Step 1: Intermediate 41

[0640]

[0641] Under argon protection, 0.2 g of N,N-dimethylglycine, 0.36 g of 1-tert-butyloxycarbonylpiperazine, 1.1 g of HATU, and 0.6 g of TEA were dissolved in 60 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then detected. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 80%.

[0642] Step 2: Intermediate 42

[0643]

[0644] Under argon protection, 0.2 g of intermediate 41 was dissolved in 10 mL of dichloromethane, and 5 mL of dioxane hydrochloride solution was added. The mixture was reacted at room temperature for 2 h, and the reaction was monitored. The solvent was evaporated and the mixture was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 90%.

[0645] Step 3: Synthesis of Example 37

[0646]

[0647] Under argon protection, 0.05 g of intermediate 42, 0.18 g of intermediate 11, 0.17 g of HATU, and 0.1 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 60%.

[0648] NMR data were as follows: 1H NMR (400MHz, CDCl3) δ 7.76 (dd, 1H), 4.40 (m, 1H), 4.18 (s, 1H), 3.95 (dt, 1H), 3.64 (t, 8H), 3.32 (m, 3H), 3.14 (d, 2H), 2.28 (d, 6H), 1.66 (m, 27H), 1.08 (dd, 3H), 0.99 (m, 6H), 0.75 (d, 3H). Mass spectrometry: [M+1] 655.6.

[0649] Example 38

[0650]

[0651] The specific preparation process is as follows:

[0652] Step 1: Intermediate 43

[0653]

[0654] Under argon protection, 0.1 g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.1 g of 2-bromo-N,N-dimethylacetamide, and 0.08 g of TEA were dissolved in 30 mL of dichloromethane and reacted at room temperature for 12 h. The reaction was then monitored. The solvent was evaporated and the solution was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric development. The yield was 80%.

[0655] Step 2: Intermediate 44

[0656]

[0657] Under argon protection, 0.1 g of intermediate 43 was dissolved in 10 mL of dichloromethane, and 5 mL of dioxane hydrochloride solution was added. The reaction was carried out at room temperature for 2 h, and the reaction was monitored. The solvent was evaporated and the mixture was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 90%.

[0658] Step 3: Synthesis of Example 38

[0659]

[0660] Under argon protection, 0.05 g of intermediate 44, 0.18 g of intermediate 11, 0.17 g of HATU, and 0.09 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 60%.

[0661] NMR data: 1H NMR (400MHz, DMSO) δ 8.18 (d, 1H), 8.12 (d, 1H), 7.58 (d, 1H), 7.43 (ddd, 2H), 7.20 (m, 3H), 6.96 (pd, 2H), 4.76 (d, 2H), 4.42 (q, 2H), 3.56 (s, 3H), 2.50 (p, 2H), 1.28 (t, 3H). Mass spectrometry: [M+1] 655.6.

[0662] Example 39

[0663]

[0664] The specific preparation process is as follows:

[0665] Step 1: Intermediate 45

[0666]

[0667] Under argon protection, 0.1 g of N,N-dimethylaminobromoethane hydrobromide, 0.08 g of tert-butyl 1,4-diazacycloheptane-1-carboxylate, and 0.13 g of TEA were dissolved in 30 mL of dichloromethane and reacted at room temperature for 12 h. The reaction was then monitored. The solvent was evaporated and the solution was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric development. The yield was 80%.

[0668] Step 2: Intermediate 46

[0669]

[0670] Under argon protection, 0.1 g of intermediate 45 was dissolved in 10 mL of dichloromethane, and 5 mL of dioxane hydrochloride solution was added. The reaction was carried out at room temperature for 2 h, and the reaction was monitored. The solvent was evaporated and the mixture was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 90%.

[0671] Step 3: Synthesis of Example 39

[0672]

[0673] Under argon protection, 0.05 g of intermediate 46, 0.18 g of intermediate 11, 0.17 g of HATU, and 0.09 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 60%.

[0674] The NMR data are as follows: 1H NMR (400MHz, CDCl3) δ 7.90 (d, 1H), 4.40 (q, 1H), 4.18 (m, 1H), 3.95 (dd, 1H), 3.7 (m, 5H), 3.32 (t, 3H), 3.10 (m, 4H), 2.85 (d, 6H), 2.35 (m, 5H), 1.15–2.10 (m, 27H), 1.08 (dd, 3H), 0.97 (m, 6H), 0.75 (d, 3H), 0.75 (s, 3H). Mass spectrometry: [M+1] 655.6.

[0675] Example 40

[0676]

[0677] The specific preparation process is as follows:

[0678] Step 1: Intermediate 47

[0679]

[0680] Under argon protection, 0.2 g imidazole, 0.5 g 1-bromo-2-chloroethane, and 0.6 g TEA were dissolved in 30 mL dichloromethane and reacted at room temperature for 12 h. The reaction was then monitored. The solvent was evaporated and the solution was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric development. The yield was 80%.

[0681] Step 2: Intermediate 48

[0682]

[0683] Under argon protection, 0.1 g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.08 g of intermediate 47, and 0.08 g of TEA were dissolved in 30 mL of dichloromethane and reacted at room temperature for 12 h. The reaction was then monitored. The solvent was evaporated and the mixture was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric development. The yield was 70%.

[0684] Step 3: Intermediate 49

[0685]

[0686] Under argon protection, 0.1 g of intermediate 48 was dissolved in 10 mL of dichloromethane, and 5 mL of dioxane hydrochloride solution was added. The mixture was reacted at room temperature for 2 h, and the reaction was monitored. The solvent was evaporated and the mixture was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 90%.

[0687] Step 4: Synthesis of Example 40

[0688]

[0689] Under argon protection, 0.05 g of intermediate 49, 0.17 g of intermediate 11, 0.16 g of HATU, and 0.08 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 60%.

[0690] The NMR data are as follows: ¹H NMR (400MHz, CDCl₃) δ 7.90 (d, ¹H), 7.62 (s, ¹H), 7.07 (s, ¹H), 6.98 (d, ¹H), 4.40 (m, ¹H), 4.05 (t, 2H), 4.18 (m, ¹H), 3.95 (dt, ¹H), 3.64 (q, 2H), 3.54 (t, 2H), 3.29 (d, 3H), 2.71 (td, 2H), 2.46 (q, 4H), 1.15–2.10 (m, 27H), 1.08 (dd, 3H), 0.97 (m, 6H), 0.75 (d, 3H). Mass spectrometry: [M+1] 664.6.

[0691] Example 41

[0692]

[0693] The specific preparation process is as follows:

[0694] Step 1: Intermediate 50

[0695]

[0696] Under argon protection, 0.2 g of 2-diethylamino-1-bromoethane hydrobromide and 0.15 g of 1-tert-butoxycarbonylpiperazine were dissolved in 40 mL of acetonitrile and reacted at 50 °C for 12 h. The reaction was then monitored. The solvent was evaporated and the solution was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 80%.

[0697] Step 2: Intermediate 51

[0698]

[0699] Under argon protection, 0.1 g of intermediate 50 was dissolved in 10 mL of dichloromethane, and 5 mL of ethyl acetate hydrochloride solution was added. The reaction was carried out at room temperature for 12 h, and the reaction was monitored. The solvent was evaporated and the solution was directly added to the next reaction step, where phosphomolybdic acid was used for colorimetric reaction. The yield was 90%.

[0700] Step 3: Synthesis of Example 41

[0701]

[0702] Under argon protection, 0.05 g of intermediate 51, 0.14 g of intermediate 11, 0.15 g of HATU, and 0.08 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and color development with phosphomolybdic acid. The yield was 60%.

[0703] NMR data are as follows: 1H NMR (400MHz, CDCl3) δ 7.32 (dd, 1H), 4.41-4.45 (m, 1H), 3.94-3.98 (m, 1H), 3.75-3.78 (m, 1H), 3.65 (t, 2H), 3.56 (t, 2H), 3.28-3.32 (m, 3H), 2.63-2.80 (m, 8H), 2.47-2.52 (m, 4H), 1.32-2.10 (m, 27H), 1.08 (dd, 3H), 0.99 (m, 6H), 0.75 (s, 3H). Mass spectrometry: [M+1] 669.7.

[0704] Example 41A, Example 41B

[0705]

[0706] The synthesis process of Example 41A is the same as in Example 41, except that intermediate 11 is replaced by intermediate 18. The resulting NMR data are as follows: 1H NMR (CDCl3-d6, 400MHz): 7.98 (d, 1H), 4.42 (q, 1H), 4.18 (s, 1H), 3.95 (dd, 1H), 3.66 (t, 2H), 3.57 (t, 2H), 3.31 (d, 1H), 3.29 (…). s,2H), 2.67-2.82(m,8H), 2.47-2.52(m,4H), 1.58-2.10(m,11H), 1.15-1.50(m,16H), 1.09-1.10(m,6H), 1.08(d,3H), 0.99(d,3H), 0.95(s,3H), 0.75(s,3H). Mass spectrometry: [M+1]669.7.

[0707] The synthesis process of Example 41B is the same as that of Example 41, except that intermediate 21 replaces intermediate 11. The resulting NMR data are as follows (CDCl3-d6, 400MHz): 7.35 (d, 1H), 4.42 (q, 1H), 3.98 (dd, 1H), 3.73-3.78 (m, 1H), 3.63 (t, 2H), 3.56 (t, 2H), 3.32 (d, 1H), 3.2 8 (s, 2H), 2.63–2.76 (m, 8H), 2.47–2.52 (m, 4H), 1.58–2.10 (m, 11H), 1.15–1.50 (m, 16H), 1.09–1.10 (m, 6H), 1.08 (d, 3H), 0.99 (d, 3H), 0.95 (s, 3H), 0.75 (s, 3H). Mass spectrometry: [M+1] 669.6.

[0708] Example 42

[0709]

[0710] The specific preparation process is as follows:

[0711] Step 1: Intermediate 52

[0712]

[0713] Under argon protection, 1 g of intermediate 3, 0.933 g of 3-(1-tert-butyloxycarbonylpiperazine-4-YL)propionic acid, 1.373 g of HATU, and 0.788 g of DIPEA were dissolved in 20 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then monitored. The mixture was extracted with water / dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), followed by TLC (DCM:MeOH = 10:1) and vanillin staining. The yield was 65%.

[0714] Step 2: Intermediate 53

[0715]

[0716] 1 gram of intermediate 52 was dissolved in 10 mL of MeOH, and 4 M HCl / dioxane was added. The reaction was allowed to proceed for 3 hours. The reaction was then monitored. After the reaction was complete, the solvent was evaporated to obtain the product, which was directly added to the next reaction step. Vanillin was used for colorimetric analysis. The yield was 80%.

[0717] Step 3: Synthesis of Example 42

[0718]

[0719] Under argon protection, 0.2 g of intermediate 53, 0.077 g of 3-pyrrolidine-1-ylpropionic acid, 0.205 g of HATU, and 0.091 g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was then detected. Extraction was performed with water / dichloromethane, followed by washing with saturated brine, drying over anhydrous sodium sulfate, and then the solvent was evaporated. The solution was purified by silica gel column chromatography (DCM:MeOH = 20:1), and the solution was developed with vanillin. The yield was 60%. NMR data were as follows: ¹H NMR (400 MHz, CDCl₃) δ 7.13 (d, 1H), 4.42 (q, 1H), 3.62 (m, 9H), 2.89 (d, 2H), 2.51 (m, 17H), 1.31 (m, 38H). Mass spectrometry: [M+1] 682.6.

[0720] Example 42A

[0721]

[0722] The preparation process was the same as in Example 42, except that intermediate 7 was used instead of intermediate 3 to obtain Example 42A. The NMR data were: 1H NMR (400MHz, CDCl3) δ 8.29 (d, 1H), 4.41 (m, 1H), 4.20 (s, 1H), 3.95 (dd, 1H), 3.55 (m, 6H), 2.83 (ddd, 2H), 2.56 (m, 16H), 1.34 (m, 40H). Mass spectrometry: [M+1] 682.7.

[0723] Example 43 Compound Activity Test

[0724] Experiment 1. Detection of the protective effect of small molecule compounds against oxygen deprivation (OGD)-induced damage in primary cultured neurons of rats.

[0725] Experimental and Analytical Methods: Pregnant rats at 15 days of gestation were ordered, and fetal brains were dissected (E15-16). Primary cortical neurons were cultured in neuronal medium in 48-well plates for 14 days (DIV 14) for oral glucose-depletion (OGD) experiments. During the OGD experiment, the neuronal culture medium was replaced with glucose-free anaerobic medium (95% N2 / 5% CO2 equilibration), and neurons were treated with OGD in the OGD chamber. Then, the medium was replaced with normal neuronal culture medium, and the cells were incubated in a 95% air / 5% CO2 incubator for 24 hours before neuronal activity analysis. Hoechst 33342 and Propidium iodide staining were performed, and high-content fluorescence microscopy was used for imaging. Twenty random, non-overlapping images were taken from each repeat well using Thermo Scientific software. TMThe total number of neurons and the number of dead neurons were counted using HCS StudioCell Analysis Software. The standard deviation and standard error were calculated using the obtained data, and a T-test analysis was performed to calculate the p-value to determine if there were any significant differences.

[0726] Compound preparation: Thirteen small molecule compounds from Examples 1 to 20 were diluted with DMSO to a stock solution of 0.5 mM. 1 μL of the stock solution was added to each well of a 48-well plate to a final concentration of 1 μM (0.5 ml of culture medium). 1 μL of DPQ (stock solution 10 mM) was added to the positive control compound and 1 μL of DMSO (negative control) was added to the culture medium 24 hours before the OGD experiment. The same concentration was added to the sugar-free and anaerobic culture medium and the subsequent normal neuronal culture medium.

[0727] Result: As Figure 1 As shown in the results of this OGD experiment, the proportion of dead cells in neurons without OGD was 10.32%, while the proportion of dead cells in the negative control with DMSO was 52.31%, with an injury rate of approximately 42%. The proportion of dead cells in the positive control with DPQ was 22.22%, demonstrating a very significant protective effect against neuronal damage.

[0728] Experiment 2. Detection of the protective effect of small molecule compounds in Examples 1 to 14B against hydrogen peroxide (H2O2)-induced oxidative damage in human SHSY5Y neurotumor cells.

[0729] Table 1. Experimental conditions and methods

[0730] cell lines SHSY5Y Plating cell state Cells in good condition Platelet cell density <![CDATA[3x10 5 [pcs / ml]]> Final drug concentration 1μM Drug protection time 24 hours Detection methods MTT: 490nm

[0731] Table 2. Experimental Procedure

[0732]

[0733] Result: As Figure 2 As shown, Examples 2, 7, 13, and 14A significantly protected against hydrogen peroxide-induced neurotumor cell damage. Among them, undifferentiated SH-SY5Y cells were pre-protected for 24 hours, with a final concentration of 1 μM, and were detected after 24 hours of hydrogen peroxide treatment.

[0734] Experiment 3. Detection of the protective effect of small molecule compounds against L-cysteine-induced neuronal excitotoxicity.

[0735] Experimental and analytical methods: Before the experiment, 24-well cell culture plates were pretreated with Poly-D-lysine and placed overnight in a cell culture incubator at 37°C and 5% CO2. SD rats at 18 days of gestation were ordered, and the fetal brains (E18-19) were dissected. Primary hippocampal neurons were cultured in 24-well plates using neuronal culture medium and serum-free medium. After culturing in a cell culture incubator at 37°C and 5% CO2 for 19 days (DIV19), the test compound (final concentration 1 μM) and the positive control AP5 [(2R)-amino-5-phosphatric acid (ester)] (final concentration 100 μM) were added. After 24 hours of culture, L-Cystine (400 μM) and NaHCO3 (10 mM) were added. After another 18 hours, hoechst3342 (2.5 μg / ml) was added for cell staining. Cells were incubated in an incubator for 15 minutes, and photographed using a high-content fluorescence microscope. Six random, non-overlapping images were taken from each repeat well. The total number of neurons and the number of dead neurons were counted using ImageJ software. The obtained data were used to determine whether there were differences between the drug-treated group and the control group.

[0736] Compound preparation: The 32 small molecule compounds from Examples 1 to 19A were diluted with DMSO to a 1 mM stock solution. 1 μl of the stock solution was added to 1 ml of culture medium in each well of a 24-well plate to a final concentration of 1 μM. 100 μl of AP5 was added as the positive control compound (100 mM stock solution), and 1 μl of DMSO was added as the negative control.

[0737] Result: As Figure 3 As shown, the proportion of dead neurons in the control group without L-Cystine (400 μM) and NaHCO3 (10 mM) was 20.12%, while the proportion of dead neurons in the negative control group with DMSO was 87.62%, with a damage rate of approximately 67.5%. The proportion of dead neurons in the positive control group with AP5 was 27.58%, indicating that AP5 had a very significant protective effect against neuronal excitotoxicity. The results of experiments with the addition of the above four small molecule compounds showed that compounds in Examples 1, 1A, 2, 4B, 6, 10, 13, and 13B had a protective effect against L-Cystine (400 μM)-induced neuronal excitotoxicity.

[0738] Experiment 4. Evaluation of the anti-inflammatory effects of "Example 5" and "Example 6" in zebrafish

[0739] laboratory animals

[0740] Transgenic neutrophil fluorescent zebrafish were bred by natural paired mating. They were 3 days post-fertilization (3 dpf) old, with a total of 810 fish, 30 fish in each experimental group. They were used to determine the maximum test concentration (MTC) in the LPS-induced inflammation experiments of "Example 5" and "Example 6", and to evaluate the anti-inflammatory effects of "Example 5" and "Example 6" on LPS-induced inflammation.

[0741] The zebrafish were raised in fish-raising water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, with a conductivity of 480 - 510 μS / cm; pH of 6.9 - 7.2; hardness of 53.7 - 71.6 mg / L CaCO3). They were provided by the fish-raising center of our company, and the experimental animal use license number was: SYXK(Zhe)2012 - 0171. The feeding management met the requirements of international AAALAC accreditation.

[0742] Experimental drugs

[0743] "Example 5", white powder, stored dry at 4°C, sampled on October 18, 2019, provided by Shenzhen Qingbo Huineng Pharmaceutical Technology Co., Ltd. Before the experiment, it was prepared into a 20 mM stock solution with DMSO and stored at -20°C.

[0744] "Example 6", white powder, stored dry at 4°C, sampled on October 18, 2019, provided by Shenzhen Qingbo Huineng Pharmaceutical Technology Co., Ltd. Before the experiment, it was prepared into a 20 mM stock solution with DMSO and stored at -20°C.

[0745] Indomethacin, batch number 1108939, produced by Shanghai Jingchun Industrial Co., Ltd., provided by Hangzhou Huante Biotechnology Co., Ltd. Before the experiment, it was dissolved and prepared into an 80 mM stock solution with DMSO and stored at -20°C.

[0746] Instruments and reagents

[0747] Dissecting microscope (SZX7, OLYMPUS, Japan); camera connected to the microscope (VertA1); precision electronic balance (CP214, OHAUS, America CP214, OHAUS); fluorescence microscope (AZ100, Nikon, Japan); methyl cellulose (Sigma, USA); dimethyl sulfoxide (Sigma, France); 6-well plate (Nest Biotech).

[0748] Concentration groups

[0749] 1. Determine the maximum test concentration (MTC) of "Example 5" and "Example 6"

[0750] Experimental group 1: Normal control group (without any treatment)

[0751] Experiment 2 Model Control Group

[0752] Experimental Group 3, "Example 5", 0.625 μM

[0753] Experimental Group 4, "Example 5", 1.25 μM

[0754] Experiment 5, "Example 5", 2.5 μM

[0755] Experiment 6, "Example 5", 5μM

[0756] Experiment 7, "Example 5", 10 μM

[0757] Experimental Group 8, "Example 5", 50 μM

[0758] Experimental Group 9, "Example 5", 100 μM

[0759] Experiment 10, "Example 5", 200μM

[0760] Experiment 11, "Example 6", 0.625 μM

[0761] Experiment 12, "Example 6", 1.25 μM

[0762] Experiment 13, "Example 6", 2.5 μM

[0763] Experiment 14, "Example 6", 5μM

[0764] Experiment 15, "Example 6", 10 μM

[0765] Experiment 16, "Example 6", 50 μM

[0766] Experiment 17, "Example 6", 100 μM

[0767] Experiment 18, "Example 6", 200 μM

[0768] 2. Evaluate the anti-inflammatory effects of "Example 5" and "Example 6".

[0769] Experiment 1: Normal control group (no treatment)

[0770] Experiment 2 Model Control Group

[0771] In the experimental group 3, the positive control drug was indomethacin 80 μM.

[0772] Experimental Group 4, "Example 5", 0.28 μM

[0773] Experimental Group 5, "Example 5", 0.83 μM

[0774] Experiment 6, "Example 5", 2.5 μM

[0775] Experiment 7, "Example 6", 0.28 μM

[0776] Experimental Group 8, "Example 6", 0.83 μM

[0777] Experimental Group 9, "Example 6", 2.5 μM

[0778] Basis for concentration determination

[0779] After communicating with the client based on the results of concentration exploration, it was determined that the maximum detection concentration for evaluating the anti-inflammatory effects of "Example 5" and "Example 6" was 2.5 μM.

[0780] Model making

[0781] LPS was used to treat normal 3dpf transgenic neutrophil fluorescent zebrafish by yolk sac injection to establish a zebrafish inflammation model.

[0782] Experimental methods

[0783] 1. Determine the maximum detection concentration (MTC) for "Example 5" and "Example 6".

[0784] Healthy, uniformly developed transgenic neutrophilic fluorescent zebrafish (3 days post-fertilization, 3dpf) were selected under a microscope and placed in six-well plates. Thirty zebrafish were randomly selected from each well, with a volume of 3 mL per well. LPS was injected into the yolk sac to establish a zebrafish inflammation model. "Example 5" and "Example 6" were administered water-soluble at concentrations of 0.625, 1.25, 2.5, 5, 10, 50, 100, and 200 μM, respectively. A normal control group (i.e., zebrafish treated with fish farming water) and a model control group were also set up. After incubation at 28°C for 3 hours, zebrafish mortality was observed and recorded. The number of dead zebrafish in each experimental group was counted to determine the maximum detectable concentration (MTC) of "Example 5" and "Example 6" for zebrafish.

[0785] 2. Evaluate the anti-inflammatory effects of "Example 5" and "Example 6".

[0786] Healthy, uniformly developed transgenic neutrophil fluorescent zebrafish (3 days post-fertilization, 3dpf) were selected under a microscope and placed in a six-well plate. Thirty zebrafish were randomly selected from each well, and the volume of each well was 3 mL. LPS was injected into the yolk sac to establish a zebrafish inflammation model. The zebrafish in "Example 5" and "Example 6" were administered water-soluble solutions at concentrations of 0.28, 0.83, and 2.5 μM, respectively; indomethacin was administered at a concentration of 80 μM. A normal control group (i.e., zebrafish treated with fish farming water) and a model control group were also established. After culturing in an incubator at 28°C for 3 hours, 10 zebrafish were randomly selected from each group for observation, photography, and image saving under a fluorescence microscope. Image analysis was performed using Nikon NIS-Elements D 3.10 advanced image processing software to calculate the number of inflammatory neutrophils (N) in the zebrafish. Statistical results were expressed as mean ± SE. The statistical analysis results of the number of inflammatory neutrophils in the zebrafish were used to evaluate whether "Example 5" and "Example 6" had significant anti-inflammatory effects on LPS-induced inflammation in zebrafish. The formulas for calculating the anti-inflammatory effects of "Example 5" and "Example 6" are as follows:

[0787] Inflammation reduction rate (%) = ((N(model control group) - N(test sample group)) / N(model control group))*100%

[0788] Statistical analysis was performed using ANOVA and Dunnett's T-test, and p < 0.05 indicated a significant difference.

[0789] Experimental results

[0790] 1. MTC

[0791] The maximum solubility of "Example 5" in DMSO is 20 mM, and the maximum DMSO concentration that zebrafish can tolerate is 1%. Therefore, the maximum detectable concentration for evaluating the anti-inflammatory effect of "Example 5" is 200 μM. At concentrations of 200, 100, 50, and 10 μM, 30 zebrafish died, with a mortality rate of 100%. At a concentration of 5 μM, 3 zebrafish died, with a mortality rate of 10%. At a concentration of 2.5 μM, the zebrafish were in good condition and no drug was precipitated. Therefore, the maximum detectable concentration for evaluating the anti-inflammatory effect of "Example 5" is 2.5 μM.

[0792] The maximum solubility of the drug in DMSO in "Example 6" is 20 mM. The maximum DMSO concentration that zebrafish can tolerate is 1%. Therefore, the maximum dosing concentration for evaluating the anti-inflammatory effect of "Example 6" is 200 μM. In "Example 6", 30 zebrafish died at concentrations of 200, 100 and 50 μM, with a mortality rate of 100%; 4 zebrafish died at concentrations of 10 and 5 μM, with a mortality rate of 13.33%; at a concentration of 2.5 μM, the zebrafish were in good condition and no drug was precipitated. Therefore, the maximum detectable concentration for evaluating the anti-inflammatory effect of "Example 6" is 2.5 μM.

[0793] The maximum detection concentrations for "Example 5" and "Example 6" were determined based on the results of concentration exploration; that is, the maximum detection concentration for evaluating the anti-inflammatory effects of "Example 5" and "Example 6" was 2.5 μM. See Table 3 for details.

[0794] Table 3. Statistics on the number of zebrafish that died at the detection concentration in "Example 5" and "Example 6" (n=30)

[0795]

[0796]

[0797] 2. Anti-inflammatory effect

[0798] As shown in Table 4, Figure 4 , Figure 5 , Figure 6 As shown, where Figure 4 The dashed area represents neutrophils at the site of inflammation. The number of neutrophils at the site of inflammation in the model control group (18) was significantly lower than that in the normal control group (3), p < 0.001, indicating the successful establishment of the LPS-induced transgenic neutrophil fluorescent zebrafish inflammation model. The number of neutrophils at the site of inflammation in the 80 μM indomethacin group (6) was significantly lower than that in the model control group, p < 0.001, with an anti-inflammatory effect of 67%, demonstrating that indomethacin has a significant anti-inflammatory effect on inflamed zebrafish.

[0799] In “Example 5”, at concentrations of 0.28, 0.83, and 2.5 μM, the number of neutrophils in the inflammatory sites of zebrafish were 12, 7, and 6, respectively, and the anti-inflammatory effects on zebrafish were 33%, 61%, and 67%, respectively. Compared with the model control group (18), the p values ​​of the 0.28, 0.83, and 2.5 μM concentration groups were all <0.001, suggesting that “Compound 3” has a significant anti-inflammatory effect on inflamed zebrafish at concentrations of 0.28–2.5 μM.

[0800] "Example 6": When the concentrations are 0.28, 0.83, and 2.5 μM, the numbers of neutrophils in the inflammatory sites of zebrafish are 12, 8, and 7 respectively, and the anti-inflammatory effects on zebrafish are 33%, 56%, and 61% respectively. Compared with the model control group (18), the p values of the 0.28, 0.83, and 2.5 μM concentration groups are all < 0.001, indicating that "Compound 4" has a significant anti-inflammatory effect on inflamed zebrafish at a concentration of 0.28 - 2.5 μM.

[0801] Table 4. Quantitative results of the effects of each experimental group on zebrafish inflammation (n = 10)

[0802]

[0803]

[0804] Experimental conclusion

[0805] Under the conditions of the experimental concentrations, both Example 5 and Example 6 have significant anti-inflammatory effects on inflamed zebrafish.

[0806] Experiment 5: Pharmacodynamic study of the compound on the rat cerebral ischemia-reperfusion (MCAO) injury model

[0807] The effects of the compound on the mouse cerebral ischemia-reperfusion (MCAO) injury model were evaluated using neurological function scores and cerebral infarction areas. The results showed that the test compound had a protective effect on mouse cerebral ischemia-reperfusion (MCAO) injury.

[0808] Experimental drugs

[0809] Table 5.待测化合物

[0810] serial number Provider batch Properties quality Storage conditions NO.1 MedChemExpress Inc. Edaravone Yellow solid powder 50mg -20 degrees Celsius (solid) NO.2 Shenzhen Qingbo Huineng Example 10 White solid powder 230mg 4 degrees NO.3 Shenzhen Qingbo Huineng Example 11 White solid powder 230mg 4 degrees NO.4 Shenzhen Qingbo Huineng Example 14A White solid powder 230mg 4 degrees NO.5 Shenzhen Qingbo Huineng Example 14B White solid powder 230mg 4 degrees NO.6 Shenzhen Qingbo Huineng Example 19A White solid powder 230mg 4 degrees

[0811] Name of normal saline: Self-made normal saline.

[0812] Name of hydroxypropyl-β-cyclodextrin: Hydroxypropyl-β-cyclodextrin; Supplier: Sigma-Aldrich Chemical Technology (Shanghai) Co., Ltd.; Batch number: FG310174; Property: White solid powder; Quantity: 30 g; Storage condition: Room temperature.

[0813] Experimental methods, experimental animals and feeding

[0814] Experimental animal strain: C57BL / 6 mice; Week age: 6 - 8; Gender: Male; Ordered animal weight: 16 - 20 g; Used animal weight: 20 - 23 g; Quantity: 40; Experimental animal provider: Zhejiang Vital River Laboratory Animal Technology Co., Ltd.; Production license number: SCKX(Zhe)2019 - 0001; Quality certificate number: No200513005, No2004280010.

[0815] Animal husbandry

[0816] Quarantine: The quarantine period is 7 days. Routine health checks are conducted by veterinarians, and animals showing abnormal behavior are removed before the experiment.

[0817] Animal housing conditions: Laboratory animals are housed in an SPF-grade, temperature- and humidity-controlled laminar flow clean room at the animal center (AAALAC certified), with three mice per cage. The room temperature is 22±3℃, humidity 40-70%, and lighting is alternating between light and dark for 12 hours. Cages: Made of polycarbonate. Soft corn cob bedding is autoclaved and sterilized, and changed twice weekly. Feed and water: Clean-grade rodent feed, purchased from Beijing Keao Xieli Feed Co., Ltd. Drinking water is autoclaved, and food is irradiated with cobalt-60 rays. Animals have free access to sterile food and water.

[0818] Animal identification: Each cage has a cage label indicating the number of animals, sex, strain, reception time, group, and experiment start time. Animal identification: Each animal is marked with a unique animal number on its tail.

[0819] Animal grouping and treatment: Based on animal weight, on day 1, the animals were randomly divided into 8 groups (N=5). One group was tested daily, with 3 animals selected from each group for cerebral blood flow measurement. Specific grouping and treatment details are shown in Table 6.

[0820] Table 6 Animal grouping and drug treatment

[0821]

[0822]

[0823] MCAO Model Making

[0824] Mice were fasted overnight before surgery, but water was permitted. They were pre-anesthetized in an isoflurane gas anesthesia machine induction chamber with an isoflurane concentration of 2.5%. After the mice showed no response when their hind paws were clamped with dissecting forceps, they were transferred to the anesthesia mask, and the isoflurane concentration was reduced to 1.5%. The mice's body temperature was maintained at approximately 37°C during surgery using a thermometer and rectal thermometer. The hair on the mice's necks was shaved while they were supine. After disinfecting the skin with iodine and alcohol, an incision was made in the midline of the neck. The tissue was bluntly dissected, and the common carotid artery (CCA) was exposed under a stereomicroscope. A ligation was made proximal to the CCA using 6-0 braided suture. The external carotid artery (ECA) and internal carotid artery (ICA) were dissected superiorly. A permanent ligation and a temporary ligation were made distal to the ECA. A loose ligation was made at the carotid sinus. The ECA vessel wall was cut at this point, and a suture plug (Beijing Xinong Technology Co., Ltd., A5-162020) was inserted. The loose ligation was then tightened to secure the suture plug. The ECA was melted using an electrocautery pen, the external carotid artery ligation was released, and a suture was inserted into the ICA until cerebral blood flow dropped to approximately 10% of baseline. The suture occluded the middle cerebral artery (MCA) for 30 minutes, after which the suture was removed, the vascular stump was cauterized, and the CCA ligation was released. The neck skin was sutured, and the mouse was placed in an intensive care cage, maintaining its postoperative body temperature at 37°C until tissue collection.

[0825] Cerebral blood flow measurement: The mouse head was fixed under stereotaxic guidance, the hair on the head was shaved, and a midline incision was made. The periosteum of the skull was removed, and the fiber optic cable of the laser Doppler flowmeter was glued to a position at Bregma AP 1.0 mm, ML 5.0 mm. Intraoperative blood flow changes in the middle cerebral artery were recorded. Successful modeling was defined as a decrease in cerebral blood flow to 80%-90% of baseline.

[0826] Drug preparation

[0827] Media: saline

[0828] Solvent: 20% hydroxypropyl-β-cyclodextrin

[0829] Preparation of PK sample: First, calculate the minimum daily drug dosage m2 based on the total weight of each group of mice and the dosage. Weigh slightly more than m2 of P2 into a bottle, and calculate the solvent volume v2 according to the drug administration volume of 10 mL / kg. Add 20% hydroxypropyl-β-cyclodextrin solution (v2) to the bottle, vortex for one minute, sonicate for 15 minutes, and vortex again for one minute until completely dissolved.

[0830] Solvent: 20% hydroxypropyl-β-cyclodextrin

[0831] Preparation of PK Samples: First, calculate the minimum daily drug dosage (m3) based on the total weight of each group of mice and the dosage. Weigh slightly more than m3 of P3 into a mortar and granulate. Calculate the solvent volume (v3) based on a drug dosage of 10 mL / kg, and measure v2 of the solvent using a syringe. Add 2-3 drops of solvent to the mortar and grind for 5 minutes. After the solvent has evaporated, add another 2-3 drops of solvent and grind for another 5 minutes, repeating this process three times. Then, wash the compound in the mortar thoroughly with solvent in small amounts several times, and add it to a suitable sample vial using a clean glass dropper. Sonicate for 15 minutes, then vortex for 5 minutes until homogeneous and free of large particles.

[0832] Detection indicators and methods

[0833] Weight: Record the weight of mice on the day before surgery and 24 hours after surgery.

[0834] Neurological function assessment: Longa behavioral scores were performed on mice 2 hours and 24 hours after surgery.

[0835] Longa behavioral scoring criteria:

[0836] 0 points: No symptoms of neurological damage;

[0837] 1 point: Unable to fully extend the opposite forepaw;

[0838] 2 points: Turn in circles to the opposite side;

[0839] 3 points: Leaning to the opposite side;

[0840] 4 points: Unable to walk on their own, loss of consciousness.

[0841] Forelimb grip strength test

[0842] Forelimb grip strength tests were performed on mice 24 hours before and 24 hours after surgery. The mice were placed on a grip strength meter and actively pulled the grip strength sensing rod. The peak grip strength was recorded. The measurements were repeated three times and the average value was taken.

[0843] Infarct area

[0844] Twenty-four hours post-surgery, mice were anesthetized with isoflurane and euthanized by decapitation. The entire brain was dissected, rinsed twice with physiological saline, and placed under a coronal facial model. The anterior 1 mm olfactory bulb and posterior 4 mm cerebellum were removed. The brain was then cut into four 2 mm slices. The brain slices were stained with 1% TTC solution at 37°C in the dark for 20 min, then transferred to 4% PFA and stored at 4°C in the dark for 48 hours before photography. The volume of cerebral infarction and edema was measured using ImageJ software.

[0845] Cerebral edema volume = (volume of the injured hemisphere - volume of the contralateral hemisphere) / volume of the contralateral hemisphere * 100%

[0846] Corrected infarct volume = (Volume of contralateral hemisphere - (Volume of injured hemisphere - Volume of white infarct area)) / Volume of contralateral hemisphere * 100%

[0847] statistics

[0848] Data from each group were analyzed using Graghpad Prism 7.0, and experimental results are expressed as mean ± standard deviation. One-way ANOVA was used to compare whether there were statistically significant differences between groups; P < 0.05 was considered statistically significant.

[0849] Experimental results

[0850] like Figure 7 As shown, during MCAO surgery, cerebral blood flow in each group (n=3) decreased to about 10% of baseline, indicating that the model was successful and could be evaluated for efficacy.

[0851] like Figure 8 As shown, the body weight of all groups decreased after surgery, but there was no significant difference in body weight among the groups of mice that underwent MCAO surgery.

[0852] like Figure 9 As shown, the grip strength of the forelimbs of mice in all groups decreased significantly after surgery. Among them, the sham-operated group and the MCAO model group showed significant differences (P<0.001), the MCAO model group and the positive control drug edaravone group showed significant differences (P<0.001), and the MCAO model group and the drug No. 3 group showed significant differences (P<0.001).

[0853] like Figure 10 As shown, the neurological deficit scores of the MCAO model group 2 hours after surgery were significantly different from those of the No. 2 and No. 3 drug groups (P<0.05); the neurological deficit scores of the MCAO model group 24 hours after surgery were significantly different from those of the positive control drug edaravone group and No. 3 drug group (P<0.05).

[0854] like Figure 11 As shown, TTC staining results indicated that the cerebral infarction volume in the MCAO model group was significantly different from that in the positive control drug edaravone group (P<0.001), drug 2 (P<0.001), drug 3 (P<0.001), drug 4 (P<0.01), and drug 5 (P<0.01).

[0855] like Figure 12 As shown, TTC staining results indicated that the cerebral edema volume in the MCAO model group was significantly different from that in the positive control drug edaravone group (P<0.01), drug 2 (P<0.001), drug 3 (P<0.001), drug 4 (P<0.001), drug 5 (P<0.05), and drug 6 (P<0.05).

[0856] Experiment 6 evaluated the effect of Example 6 in an LPS-induced mouse depression-like model.

[0857] LPS was used to induce peripheral and central inflammation in mice, thereby causing depressive-like symptoms. Changes in depression-related behavioral indicators in mice after drug administration were detected. The results showed that Example 6 (ND) had an antidepressant effect in mice.

[0858] Table 7. Compounds to be tested

[0859] serial number Compound Name Provider Properties Storage conditions 1 Example 6 (ND) Shenzhen Qingbo Huineng White solid powder 4℃

[0860] Solvent: Sodium carboxymethyl cellulose (NC); Name: Sodium carboxymethyl cellulose; Supplier: Merrill; Batch: 69881020; Appearance: White solid powder; Quantity: 200g; Storage conditions: Room temperature

[0861] Peripheral and Central Inflammatory Inducer (LPS) Name: Lipopolysaccharide; Manufacturer: Sigma; Batch: L2880-100MG; Appearance: White powder; Purity: >97%; Specification: 100mg; Storage conditions: 2-8℃, protected from light

[0862] Experimental methods, laboratory animals, and husbandry management

[0863] Laboratory animal strain: C57BL / 6J mouse; age: 6-8 weeks; sex: male; animal weight: 18-20g; quantity: 24; laboratory animal provider: Guangdong Provincial Medical Laboratory Animal Center

[0864] Animal husbandry

[0865] Quarantine: The quarantine period is 5 days. Animals showing abnormal behavior are removed before the experiment.

[0866] Animal housing conditions: Laboratory animals are housed in a clean, temperature- and humidity-controlled laminar flow room at the Animal Center of Peking University Shenzhen Graduate School, with 4-5 animals per cage. The room temperature is 22±3℃, humidity is 40-70%, and lighting alternates between light and dark 12 hours a day. Feed and water: Clean-grade rodent feed, purchased from Beijing Keao Xieli Feed Co., Ltd. Drinking water is autoclaved. Animals have free access to sterile food and water.

[0867] Animal identification numbers: Each cage has a cage label indicating the number of animals, sex, strain, reception time, group, and experiment start time. Animal identification numbers: Each animal is marked with a unique animal number on its tail.

[0868] Animal grouping and treatment: The animals were randomly divided into 3 groups. The specific grouping and treatment are shown in Table 8.

[0869] Table 8 Animal grouping and drug treatment

[0870]

[0871] Establishment of LPS-induced depression-like model

[0872] Modeling method: LPS (2 mg / kg, ip, QD) was injected into mice on days 1, 2, and 3 to induce peripheral and central inflammation. The ND drug test group was given pre-protection 2 hours before LPS injection (ND 30 mg / kg, po, QD), and forced swimming, tail suspension, and sucrose preference tests were performed on day 4.

[0873] Preparation of test drugs and model inducers

[0874] Solvent (0.5% CMC-Na solution): Take 0.5g of white CMC-Na solid powder and add it to 100ml of double-distilled water. Vortex until dissolved. The prepared solution should be stored in a sealed container at 4°C. This solution can be stored for one month, but if mold is found, it must not be used in experiments and must be prepared again.

[0875] During the experiment, the test compounds and model inducers were prepared fresh for each use. The specific preparation methods are shown in Table 9.

[0876] Table 9. Preparation of Solvents and Test Reagents

[0877]

[0878] Detection indicators and methods

[0879] General observation involves observing and recording the animal's living conditions.

[0880] Forced swim test (FST)

[0881] Mice were placed in a transparent glass cylinder (diameter: 23 cm; height: 31 cm) containing 15 cm of water, with the temperature maintained at 24 ± 1 degrees Celsius. The free-time test (FST) lasted 6 minutes, during which a high-definition camera was used to record the process. Professional testing software calculated the immobility time of the mice. After the test, the mice were immediately returned to their cages, and care was taken to keep them warm.

[0882] Sugar water preference test (SPT)

[0883] Before testing, mice were trained by placing two bottles of 1% (w / v) sucrose solution in each cage. After 24 hours, one of the bottles was replaced with pure water. After acclimatization, mice were fasted and deprived of water for 10-24 hours before testing. Two pre-weighed water bottles were placed in each cage, one containing 1% (w / v) sucrose solution and the other containing pure water. After 12 hours, both bottles were weighed again, and the consumption of sucrose solution and pure water by each mouse was recorded.

[0884] Sugar water preference index % = Sugar water consumption / (Sugar water consumption + Pure water consumption) × 100%.

[0885] Tail suspension test (TST)

[0886] After acclimatization, the mice were attached to a suspension bar with their tails attached, their heads approximately 20-25 cm above the ground, for about 6 minutes. A high-definition camera recorded the event, and behavioral analysis software was used to identify and count the time the mice remained still. The mice were returned to their cages after the experiment.

[0887] statistics

[0888] Experimental results are expressed as mean ± standard deviation. One-way ANOVA was used to compare whether there were statistical differences between groups.

[0889] Experimental results

[0890] FST

[0891] like Figure 13 As shown, the FST immobility time results for each group of mice indicated that the cumulative immobility time in Example 6 (ND) was significantly lower than that in the LPS group. **, P<0.01; ***, P<0.001.

[0892] SPT

[0893] like Figure 14 As shown, the results of the sugar water preference rate in the SPT of mice in each group indicated that Example 6 (ND) did not show a significant increase compared to the LPS group. *, P<0.05; ***, P<0.001

[0894] TST

[0895] like Figure 15 As shown, the TST immobility time results of each group of mice indicated that the cumulative immobility time in Example 6 (ND) was significantly lower than that in the LPS group. *, P<0.05; ***, P<0.001

[0896] Conclusion: Compound Example 6 (ND) showed good antidepressant effects in FST and TST experiments.

[0897] Experiment 7. Evaluation of the effect of compound Example 20A in LPS-induced mouse peripheral and central inflammation models.

[0898] LPS was used to induce peripheral and central inflammation in mice, and changes in inflammation and oxidative stress levels in serum and hippocampus were detected after drug administration. The results showed that Example 20A (ND) had a significant anti-inflammatory effect.

[0899] Test sample

[0900] Table 10 Test Compounds

[0901] serial number Compound Name Provider Properties Storage conditions 1 Example 20A(ND) Shenzhen Qingbo Huineng White solid powder 4℃

[0902] solvent

[0903] Sodium carboxymethyl cellulose (NC), Name: Sodium carboxymethyl cellulose; Supplier: Merrill; Batch: 69881020; Appearance: White solid powder; Quantity: 200g; Storage conditions: Room temperature.

[0904] Peripheral and central inflammatory inducer (LPS), Name: Lipopolysaccharide; Manufacturer: Sigma; Batch: L2880-100MG; Appearance: White powder; Purity: >97%; Specification: 100mg; Storage conditions: 2-8℃, protected from light.

[0905] Test methods

[0906] Laboratory animals and their husbandry

[0907] Laboratory animal strain: C57BL / 6J mice; age: 6-8 weeks; sex: male; animal weight: 18-20g; quantity: 24. Laboratory animal provider: Guangdong Provincial Medical Laboratory Animal Center.

[0908] Animal husbandry

[0909] Quarantine, with a quarantine period of 5 days, and animals exhibiting abnormal behavior are removed before the experiment.

[0910] Animal housing conditions: Laboratory animals are housed in a clean, temperature- and humidity-controlled laminar flow room at the Animal Center of Peking University Shenzhen Graduate School, with 4-5 animals per cage. The room temperature is 22±3℃, humidity is 40-70%, and lighting alternates between light and dark 12 hours a day. Feed and water: Clean-grade rodent feed, purchased from Beijing Keao Xieli Feed Co., Ltd. Drinking water is autoclaved. Animals have free access to sterile food and water.

[0911] Animal identification numbers: Each cage has a cage label indicating the number of animals, sex, strain, reception time, group, and experiment start time. Animal identification numbers: Each animal is marked with a unique animal number on its tail.

[0912] Animal grouping and treatment

[0913] The animals were randomly divided into 3 groups, and the specific grouping and treatment are shown in Table 11.

[0914] Table 11 Animal grouping and drug treatment

[0915]

[0916] LPS Inflammation Model Establishment

[0917] Modeling method: LPS (2 mg / kg, ip, QD) was injected into mice on days 1, 2, and 3 to induce peripheral and central inflammation. The ND drug test group was given the drug 2 hours before LPS injection for pre-protection (ND 30 mg / kg, po, QD). After the experiment, plasma and brain tissue of mice were collected for the analysis of inflammatory factors and peroxidases.

[0918] Preparation of test drugs and model inducers

[0919] Solvent (0.5% CMC-Na solution): Take 0.5g of white CMC-Na solid powder and add it to 100ml of double-distilled water. Vortex until dissolved. The prepared solution should be stored in a sealed container at 4°C. This solution can be stored for one month, but if mold is found, it must not be used in experiments and must be prepared again.

[0920] During the experiment, the test compounds and model inducers were prepared fresh for each use. The specific preparation methods are shown in Table 12.

[0921] Table 12: Preparation of Solvents and Test Reagents

[0922]

[0923] Detection indicators and methods

[0924] General observation involves observing and recording the animal's living conditions.

[0925] Animal-based

[0926] Serum: Mice were anesthetized with 1% sodium pentobarbital, and blood was collected by enucleation into EP tubes. The tubes were incubated at 4°C for 1 hour and then centrifuged at low temperature. The supernatant was collected and then frozen in liquid nitrogen.

[0927] Hippocampal tissue: Mice were anesthetized with 1% sodium pentobarbital and then perfused with cold PBS. The brain was then removed and the tissue was frozen in liquid nitrogen in EP tubes on ice.

[0928] Measurement of oxidative stress-related indicators

[0929] Frozen plasma was collected and tested for various indicators using reactive oxygen species (ROS) detection kits, hydrogen peroxide detection kits, and nitric oxide detection kits. The specific operating procedures are the same as the instructions for each kit.

[0930] Frozen hippocampal tissue was collected, and RIPA tissue lysis buffer containing protease inhibitors was added. The tissues were thoroughly homogenized and lysed. After low-temperature centrifugation, the supernatant was collected, and protein quantification was performed using a BCA protein quantification kit. All tissue samples were adjusted to a uniform protein concentration, and various indicators were measured using reactive oxygen species (ROS), hydrogen peroxide, and nitric oxide (NO) detection kits. Specific operating procedures are the same as those specified in the kit instructions.

[0931] Cytokine detection

[0932] Frozen plasma was collected and various indicators were detected using IL-1β, IL-6, and IL-10 kits. The specific operating procedures are the same as those in the instructions for each kit.

[0933] Take the supernatant of the above hippocampal tissue and use IL-1β, IL-6 and IL-10 kits to detect various indicators. The specific operation procedure is the same as the instructions of each kit.

[0934] statistics

[0935] Experimental results are expressed as mean ± standard deviation. One-way ANOVA was used to compare whether there were statistical differences between groups.

[0936] Experimental results

[0937] Oxidative stress related indicators

[0938] like Figures 16-23 As shown, the results of detecting key indicators of oxidative stress in the serum and hippocampus of mice in each group showed that the ROS level, H2O2 concentration, and NO concentration in the serum of Example 20A (ND) were significantly improved compared with those in the LPS group, indicating that Example 20A (ND) has a good antioxidant effect. *, P<0.05; **, P<0.01.

[0939] Inflammatory factor markers

[0940] like Figures 24-29 As shown, the results of the detection of important inflammation-related indicators in the serum and hippocampal tissue of mice in each group indicated that Example 20A only affected IL-1β in serum and IL-10 in the hippocampus, suggesting that Example 20A (ND) has an anti-inflammatory effect. *, P<0.05; **, P<0.01.

[0941] Experiment 8: Determination of the binding strength of small molecule compounds to the mitochondrial respiratory chain supercomplex.

[0942] Experimental and Analytical Methods: HEK293 cells were cultured under 5% CO2 conditions, and the mitochondrial respiratory chain supercomplex I1III2IV1 was purified from them. The supercomplex was chemically covalently coupled to the surface of a metal chip, and solutions of small molecules at different concentrations were passed through the chip. Changes in the reflectance of the metal surface were monitored using SPR technology on a Biacore 8K plus analyzer, and corresponding response curves for small molecule binding were plotted. The binding strength (KD value) between the small molecules and the human mitochondrial respiratory chain supercomplex I1III2IV1 was calculated using the built-in fitting software of the Biacore 8K plus instrument.

[0943] Compound preparation: Small molecule compounds from Examples 1A, 13B, 23, 24, 25, and 26 were dissolved in ddH2O to prepare 10 mM stock solutions. The Example 13B stock solution was then serially diluted with 10 mM HEPES (pH=7.4) solution to 1.5625 µM, 0.78125 µM, 0.390625 µM, and 0.1953125 µM. Similarly, the Example 1A stock solution was serially diluted with 10 mM HEPES (pH=7.4) solution to 6.25 µM, 3.125 µM, 1.5625 µM, 0.78125 µM, 0.390625 µM, and 0.1953125 µM. For measurement, 200 µL of each concentration of small molecule solution was passed through the surface of the metal chip.

[0944] The results are as follows Figures 30a to 32b As shown, the binding strength of small molecules to respiratory chain complex I reached a very high level. Experiment 9. Detection of the catalytic activity of small molecule compounds promoting SMP (submitochondrial particles).

[0945] Experimental and Analytical Methods: HEK293 cells were cultured under 5% CO2 conditions, and mitochondria were purified from them. SMPs (submitochondrial particles) were obtained by sonication of the mitochondria. SMPs were incubated with different concentrations of small molecule compounds or a control drug (Rotenone, 2 μM) for 10 minutes, followed by the addition of 500 μM NADH. NADH absorbance was measured using an Enspire multimode microplate reader, and enzyme activity curves showing the decrease in NADH concentration catalyzed by SMP were plotted. Linear fitting was performed, and the maximum reaction rate of SMP was calculated. The addition of 1 mM FeCN to the normal NADH catalytic system of SMP caused a short circuit in the electron pathway from NADH to coenzyme Q. In the presence of FeCN and Rotenone, the NADH concentration decrease curve was also measured, and linear fitting was performed to calculate the maximum reaction rate at the flavin site of SMP.

[0946] Compound preparation: Small molecule compounds from Examples 25A, 26A, 41A, 27A, 42A, 29A, 32A, 33A, 34A, and 35A were dissolved in ddH2O to prepare 10 mM stock solutions. Then, the small molecules were serially diluted with 10 mM Tris buffer to 0.075, 0.1, 0.133, 0.178, 0.237, 0.316, 0.422, 0.563, 0.75, and 1 µM for SMP catalysis experiments.

[0947] The results are as follows Figure 33 As shown, in the catalytic experimental system, the control group exhibited catalytic activity, and Rotenone completely blocked the dehydrogenation reaction of NADH, indicating that the reaction system was effective. Small molecule compounds within 1 µM (Examples 25A, 26A, 41A, 27A, 42A, 29A, 32A, 33A, 34A, and 35A) had no significant effect on the activity of SMP. However, high concentrations of small molecule compounds (Examples 25A, 26A, 41A, 27A, 42A, 29A, 32A, 33A, 34A, and 35A) had a significant impact on the activity of SMP.

[0948] Experiment 10. Detection of small molecule compounds promoting mitochondrial stress in HPAEC cells.

[0949] Experimental and analytical methods: HPAEC cells (human pulmonary artery endothelial cells) were purchased from ATCC and passaged four times under 5% CO2 conditions before being transferred to Seahorse cell culture plates, approximately 20,000 cells per well. After cell attachment, HPAEC cells were incubated with 0.5 and 1 μM of Example 13B for 48 hours. The oxygen consumption rate (OCR) of each well was measured using a Seahorse XFe96 analyzer, and OCR curves were plotted. After the measurement, the total protein content in each well was determined using BCA, and the OCR curves were corrected.

[0950] The results are as follows Figure 34As shown, in the resting state (time: 0-20 minutes), the five compounds had almost no effect on cellular oxygen consumption. The addition of oligomycin, an inhibitor of complex V, prevented the consumption of the proton gradient; the high proton gradient inhibited the activity of complex I, leading to a decrease in oxygen consumption rate, but the reduction was almost identical between the different small molecules and the control group. The addition of the uncoupling agent FCCP caused a sharp drop in the proton gradient and a significant increase in oxygen consumption. All small molecules increased the rate of increase in oxygen consumption, reflecting that these small molecules can enhance the sensitivity of complex I to the sharp drop in the proton gradient, prompting complex I to more strongly increase the electron transport rate. Finally, the addition of Rotenone (an inhibitor of mitochondrial electron transport chain complex I) and Antimycin (an inhibitor of mitochondrial electron transport chain complex III) completely blocked electron transport, reducing the oxygen consumption rate to its lowest value; the small molecules had no effect on blocking electron transport.

[0951] Experiment 11. Detection of small molecule compounds protecting HPAEC cells from SARS-CoV-2 Spike-induced ROS production.

[0952] Experimental and analytical methods: HPAEC cells (human pulmonary artery endothelial cells) were purchased from ATCC and passaged four times under 5% CO2 conditions before being transferred to MatTek cell imaging dishes, approximately 50,000 cells per dish. HPAEC cells were incubated with Spike (SARS-CoV-2 spike protein, 8 μg / mL) for 24 hours, or pre-incubated with Example 13B (0.25, 0.5, 1 μM) for 6 hours before incubating with Spike (8 μg / mL) for 24 hours. Mitochondria were then stained using MitoSox Red, PKMDR, and MitotrackerGreen mitochondrial fluorescent probes, and cell fluorescence confocal images were captured using a Leica stimulated emission depletion super-resolution confocal fluorescence microscope (STED). Subsequently, ImageJ was used to measure the fluorescence intensity of cells in different groups. The standard deviation and standard error were calculated using the obtained data, and a T-test analysis was performed to calculate the p-value to determine whether there were significant differences.

[0953] Results: After Spike was added to HPAEC cells, mitochondrial ROS was significantly increased compared to the control group (brighter red fluorescence). Figures 35a to 35b As shown, after protecting mitochondria with small molecule compounds in Examples 13B, 33, and 34, the addition of Spike resulted in a significant decrease in mitochondrial ROS. Using the PKMDR / MTG value as an indicator of the transmembrane potential difference per unit mass of mitochondria, small molecule compounds in Examples 13B, 33, and 34 showed a protective effect on the mitochondrial transmembrane potential difference.

[0954] Experiment 12. Detection of the effect of small molecule compounds on improving atherosclerosis in APOE mice

[0955] Experimental and Analytical Methods: Six-week-old male APOE- / - mutant B6J mice and WT B6J mice were purchased and acclimatized in the housing for two weeks. At eight weeks of age, all groups except the Vehicle and WT groups were fed a high-fat diet (Research Diets, D12492). At nine weeks of age, the Control group was given a 10% cyclodextrin solution, while the other groups were administered different concentrations of small molecule compounds orally every two days. After one month of treatment, approximately 500 mL of blood was collected from the orbital vein of the APOE mice. After centrifugation, the supernatant plasma was collected and sent for analysis of total cholesterol (CHOL), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels. After 16 weeks of high-fat feeding, the APOE mice were sacrificed, and the aortic arch was dissected for sampling. Mouse aortic arches were fixed with 4% paraformaldehyde, and then the internal arterial plaques of the aortic arch were stained with Oil Red chromatograph. The stained aortic arch was then imaged using a Zeiss stereomicroscope. The images were analyzed using ImageJ to determine the overall area of ​​the longitudinal section of the aortic arch and the area of ​​the orange-red plaques, calculate the plaque area ratio, and perform a T-test to calculate the p-value to determine whether there were significant differences.

[0956] Compound treatment: Small molecule compounds from Examples 13B, 37, 38, 39, and 40 ( Figures 36a to 36d The solution was added to a 10% cyclodextrin solution to achieve a final concentration of 12 mg / mL. Then, it was serially diluted with 10% cyclodextrin solution to 6, 3, and 1.5 mg / mL.

[0957] The results are as follows Figures 36a to 36dAs shown, the total cholesterol level in the serum of APOE mice in the Control group was close to 800 mg / dL after high-fat feeding, while the total cholesterol level in the serum of WT genotype mice was only about 100 mg / dL. Administration of small molecule compounds (Examples 13B, 37, 38, 39, and 40) all reduced the total cholesterol level in APOE mice. Meanwhile, small molecule compounds (Examples 13B, 37, 38, 39, and 40) had virtually no effect on high-density lipoprotein (HDL) in APOE mice, while the trend of low-density lipoprotein (LDL) was almost identical to that of total cholesterol. This indicates that small molecule compounds (Examples 13B, 37, 38, 39, and 40) primarily reduced harmful LDL while maintaining beneficial HDL. Oil Red staining of the APOE aortic arch clearly showed that the orange-red plaque area in the treatment group was significantly less than that in the Control group, which was statistically significant. The above results demonstrate that the small molecule compounds in Examples 13B, 37, 38, 39, and 40 can reduce serum LDL levels, thereby inhibiting the occurrence of atherosclerosis.

[0958] Experiment 13. Water Maze in AD Rats

[0959] Experimental and Analytical Methods: Human APP mutant protein was introduced into SD rat embryos using CRISPR Cas9 technology to obtain a transgenic AD rat model. This strain of rats showed amyloid protein deposition in the brain starting at 2 months of age and exhibited certain behavioral impairments at 5 months of age. To verify the treatment effect, medication was administered from 7 months of age to 13 months of age, and a water maze test was performed to verify the improvement in spatial memory. The water maze test consisted of three phases: Phase 1 (1 day): Adaptation phase. A 15cm diameter escape platform was placed in a 1.8m diameter pool. Four spatial markers (circles, triangles, squares, and crosses) were placed on the pool walls to indicate the cardinal directions. Rats were sequentially placed into the clear water along the pool walls, with the water level 1cm below the escape platform. The rats easily found the escape platform, climbed onto it, and were rescued from the water maze, thus learning the basic rules of the water maze. Phase 2 (4 days): Training phase. The pool setup remains unchanged, but the water level is raised 1 cm above the escape platform, and ink is poured in to turn the water black, making the escape platform invisible. Each day, rats are placed into the pool from the four cardinal directions (east, south, west, and north). They are rescued after successfully climbing onto the escape platform. If the rat cannot find the platform within 90 seconds, it is guided to climb onto it and stand for 15 seconds before being rescued. The third stage, lasting one day, is the testing stage. The escape platform is removed, and the rat is placed back in along the pool wall. Rats with strong spatial memory will repeatedly navigate to the original escape platform location. The latency to the platform upon first arrival, the number of times the rat traverses the platform, the average distance to the platform, and the time spent in the target quadrant are recorded.

[0960] Results: A total of three groups of mice were included: the WT group (n=13), the model group (AD group) (n=7), and the drug-treated group (Example 13B group) (n=11). The latency period for the first arrival at the plateau was as follows: Figure 37a ), number of platform visits (e.g.) Figure 37b ), and the average distance from the platform (e.g. Figure 37c ), the time spent in the target quadrant (e.g.) Figure 37d In terms of indicators such as ( ), the rats in the drug-treated group showed improved behavioral performance compared to the model group. Figures 37e to 37g The display shows heatmaps of the movement trajectories of rats in each group in the pool.

[0961] Experiment 14. T-maze of AD rats

[0962] Experimental and Analytical Methods: Human APP mutant protein was introduced into SD rat embryos using CRISPR Cas9 technology to obtain a transgenic AD rat model. This strain of rats showed amyloid protein deposition in the brain starting at 2 months of age and exhibited certain behavioral impairments at 5 months of age. To verify the therapeutic effect, medication was administered from 7 months of age to 15 months of age, and a T-maze test was performed to verify its improvement on working memory. The T-maze has four phases: I. Adaptation Phase: Food is placed in both arms, and both valves are open. Each rat is trained twice. The next phase begins when all rats have consumed food from both sides within 5 minutes in both tests. II. Forced Selection Phase: One valve is closed, allowing the rat to consume food from the opposite side. Then, the opposite valve is closed again, allowing the rat to consume food from the other side. This constitutes one training session. Each animal is trained four times daily for three days. III. Self-Selection Phase: One valve is partially closed, allowing the rat to consume food from the opposite side. Then, all valves are opened; if the rat moves to the other side, it can consume the food there. If the rat moves to the same side, it receives no food and is confined to that side for 30 seconds. This constitutes one training session. Each animal is trained four times per day until the accuracy rate exceeds 75% for two consecutive days, at which point the animal proceeds to the next stage. IV. Delayed Self-Selection Test Stage: This stage is essentially the same as Stage III, except that an interval is added before the self-selection begins after the forced selection ends. The intervals are set to 1.5 min, 3 min, and 10 min. Each interval is tested four times.

[0963] Results: A total of three groups of mice were included: WT group (n=3), model group (AD group) (n=2), and drug-treated group (Example 13B group) (n=6). Figure 38 As shown, there was no significant difference between the model group and the drug-treated group at 1.5 min and 3 min, but at 10 min, the working memory of the model group was significantly weakened, while the working memory of the drug-treated group remained intact.

[0964] Experiment 15. Nesting behavior in AD mice

[0965] Experimental and Analytical Methods: This experiment used transgenic mice introduced in Example 13B of APP / P, with C57BL / 6J as the background mice. The phenotype of this model mouse was characterized by cognitive behavioral changes at 3 months of age, the appearance of senile plaques at 5 months of age, and the formation of numerous senile plaques at 12 months of age. One symptom of Alzheimer's disease (AD) is inability to care for oneself, and nesting behavior reflects the self-care level of mice. At the start of the experiment, 10 rectangular pieces of paper were neatly arranged in order in the mouse cages. Normal mice would tear the paper into pieces and build a nest, while mice with severe AD symptoms would not tear the paper to build a nest. Scores were given based on the degree of tearing and the completeness of the nest; higher scores indicated better nesting behavior and a higher level of self-care.

[0966] Results: In this experiment, medication was administered starting at 2 months of age. Nesting behavior scoring was performed at 3 and 6 months of medication. Four small molecules were tested, as described in Examples 23, 24, 25, and 26. Results are as follows... Figure 39a and 39b The results showed that the nest-building behavior of the model group was significantly reduced compared to the WT group, and Example 25 showed the most significant improvement in nest-building behavior.

[0967] Experiment 16. Water Maze in AD Mice

[0968] Experimental and Analytical Methods: This experiment used transgenic mice introduced in Example 13B of APP / P, with C57BL / 6J as the background mice. The phenotype of this model mouse was characterized by cognitive behavioral changes at 3 months of age, the appearance of senile plaques at 5 months of age, and the formation of numerous senile plaques at 12 months of age. The water maze experiment was divided into three stages: Stage 1 (1 day): Adaptation stage. An 8cm diameter escape platform was placed in a 1.2m diameter pool. Four spatial markers (circles, triangles, squares, and crosses) were placed on the pool walls to indicate the four cardinal directions. Rats were sequentially placed into the clear water along the pool walls, with the water level 1cm below the escape platform. The mice easily found the escape platform, climbed it, and were rescued from the water maze, thus learning the basic rules of the water maze. Stage 2 (4 days): Training stage. The pool setup remained unchanged, but the water level was raised to 1cm above the escape platform, and ink was poured in to make the water black, obscuring the escape platform. Each day, mice were placed into the pool from the four cardinal directions and rescued after successfully climbing the escape platform. If the platform is not found within 90 seconds, the rat is guided to climb onto it and stand for 15 seconds before being rescued. The third stage, lasting one day, is the testing stage. The escape platform is removed, and the rat is placed back in along the pool wall. Rats with strong spatial memory will repeatedly move between the original escape platform location. The number of platform movements and the time spent in the target quadrant are recorded.

[0969] Results: In this experiment, medication was administered starting at 2 months of age. Spatial memory was assessed using the water maze test 6 months after administration. Four small molecules were tested in Examples 23, 24, 25, and 26. Results are as follows: Figure 40a and 40b The results show that the spatial memory ability of the model group is significantly weakened compared to the WT group, while the improvement of spatial memory ability in Example 25 is the most obvious in terms of indicators such as the number of platform shuttles and the dwell time in the target quadrant.

[0970] Experiment 17. Light and Dark Box for AD Mice

[0971] Experimental and Analytical Methods: This experiment used transgenic mice introduced in Example 13B of APP / P, with C57BL / 6J as the background mice. The phenotype of this model mouse was cognitive behavioral changes at 3 months of age, the appearance of senile plaques at 5 months of age, and the formation of numerous senile plaques at 12 months of age. The principle of the light-dark box experiment is that mice have a natural tendency to move towards darkness; when placed in a light box, they will spontaneously enter the connected dark box. However, in this experiment, the dark box was equipped with an electric stimulation mechanism, and the mice would be shocked upon entering. Thus, normal mice stopped entering the dark box after being shocked several times, while AD mice did not remember being shocked upon entering and would continue to enter. Therefore, the latency period before the mice spontaneously enter the dark box after being placed in the light box reflects the strength of the mouse's memory of the electric shock; the later the mice enter the dark box, the stronger their memory of the electric shock.

[0972] Results: In this experiment, medication was administered starting at 2 months of age. Spatial memory was assessed using the water maze test 6 months after administration. Four small molecules were tested in Examples 23, 24, 25, and 26. Results are as follows: Figure 41 The results showed that the model group had a significantly weaker memory of electric shock compared to the WT group, while the two small molecules in Examples 23 and 25 could effectively enhance the model mice's memory of electric shock.

[0973] Experiment 18. TDP43 A315T mouse lifespan

[0974] Experimental and analytical methods: This experiment used TDP43 A315T Transgenic mice, a strain that mimics the pathogenesis of ALS (Amyotrophic Lateral Sclerosis). These mice typically begin to die around day 90, reaching a median mortality rate around day 120. Our experiment consisted of two groups: a model group and a treatment group. The model group received no treatment, while the treatment group received the drug starting on day 60, testing the small molecule Example 13B at a dose of 40 mpk. Each group contained 20 mice. The date of death and body weight of each mouse were recorded, and body mass curves and survival curves were plotted.

[0975] Results: Body weight is an important indicator of ALS disease progression. Figure 42aAs shown, the body weight of the model group mice began to decrease significantly at week 13, while the weight of the drug-treated group mice did not begin to decrease significantly until week 16. Figure 42b The survival curves shown indicate that the model group mice began to die en masse from day 90, with a median mortality rate of approximately 120 days, and all mice died by day 140. In contrast, the drug-treated group only began to die after day 100, with mass mortality starting after day 120, a median mortality rate of 135 days, and all mice died by day 150. These results demonstrate that the small molecule Example 13B can effectively prolong TDP43. A315T The survival time of transgenic mice suggests that this small molecule may effectively prolong the survival time of ALS patients.

[0976] Experiment 19. SOD G93A Mouse gait analysis

[0977] Experimental and analytical methods: This experiment used SOD. G93A Transgenic mice, this strain of mice mimics the pathogenesis of ALS (Amyotrophic Lateral Sclerosis), exhibiting significant motor dysfunction with increasing age. Gait analysis is a behavioral analysis method for assessing motor dysfunction. Mice with motor dysfunction show a significantly reduced stride length. Our experiment consisted of two groups: a model group and a drug-treated group. The model group received no drug, while the drug-treated group received the drug starting on day 60, testing for the small molecule Example 13B at a dose of 40 mpk. Ten mice were used in each group, and the stride length of each mouse was measured at week 19.

[0978] result: Figure 43 The results showed that the stride length of the mice in the drug-treated group was much greater than that of the mice in the model group, indicating that the small molecule Example 13B significantly improved the motor ability of SODG93A mice.

[0979] Experiment 20. SOD G93A Mouse open field

[0980] Experimental and analytical methods: This experiment used SOD. G93A Transgenic mice, a strain that mimics the pathogenesis of ALS (Amyotrophic Lateral Sclerosis), exhibit significant motor dysfunction with increasing age. Calculating average movement speed in an open field is a behavioral analysis method for assessing motor dysfunction. Mice with motor dysfunction show a significantly reduced average movement speed. Our experiment consisted of three groups: a WT group, a model group, and a drug-treated group. The WT group consisted of normal mice, the model group received no drug, and the drug-treated group received the drug starting on day 60, testing for the small molecule Example 13B at a dose of 40 mpk. Ten mice were in each group, and the experiment was conducted weekly from week 14 until week 19.

[0981] result: Figure 44The results showed that, over the 6 weeks of testing, the average movement rate of mice in the drug-treated group was significantly higher than that of mice in the model group, indicating that the small molecule Example 13B significantly improved SOD levels. G93A The motor ability of mice.

[0982] Experiment 21. DSS mouse enteritis model

[0983] Experimental and Analytical Methods: The purpose of this experiment was to investigate whether the test substances (Examples 13B, 33, and 34) administered orally multiple times via gavage had a therapeutic effect on ulcerative colitis induced by 3.0% sodium dextran sulfate (DSS) in mice. Sixty healthy mice were randomly divided into six groups of ten mice each, based on their body weight: a normal control group, a model group, a positive control group, and groups containing different types of test substances (Examples 13B, 33, and 34, all at 80 mpk). Mice other than the normal control group were given free access to 3.0% DSS solution for 8 consecutive days to induce the ulcerative colitis model. The normal control group was given free access to autoclaved filtered water. Drug administration began on the day of model initiation, with the test substances administered orally via gavage at the designed dose once daily for 9 consecutive days. During the administration period, the mice's body weight was monitored daily, and the shape of their feces and the presence of bleeding were observed. The day after the last administration, following deep CO2 anesthesia, blood was drawn from the heart, and plasma was collected and stored at -80°C. Subsequently, the abdominal cavity was opened, the liver was removed, and stored at -80°C. Finally, the entire colon was harvested, its length was measured, photographed, and after being dissected along the mesenteric side, the colonic contents were washed with physiological saline and weighed. A portion of the lesion tissue was fixed in 10% formalin and subjected to HE staining for histopathological observation; the remaining colonic tissue was stored at -80°C.

[0984] Result: (1) As Figure 45a As shown, the effects on the general condition of DSS colitis mice: Four days after 3.0% DSS modeling, the model animals successively showed abnormalities such as loose stools, bloody stools, and weight loss, which gradually worsened thereafter; the model group mice also showed symptoms of reduced appetite, fatigue, emaciation, piloerection, and dull hair. 720mg / kg of the positive drug SASP and the test substance (Example 13B, Example 33, Example 34) both had certain ameliorative effects, but by the experimental endpoint, weight loss and abnormalities such as loose stools and bloody stools still occurred. (2) As Figure 45b As shown, the effects on colon weight and length in DSS colitis mice were as follows: After free access to 3.0% DSS solution, the colon of mice showed atrophy, which was manifested as shortening of colon length and reduction in weight; both the positive control drug and the test substance could inhibit colon atrophy in mice; the test substances (Examples 13B, 33, and 34) could increase the colon weight and length of DSS mice and inhibit colon atrophy in mice.

[0985] Experiment 22. TNBS rat enteritis model

[0986] Experimental and Analytical Methods: The purpose of this experiment was to investigate whether repeated oral gavage administration of the test substance (Examples 13B, 36, and 37, all at 80 mpk) had a therapeutic effect on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ulcerative colitis in rats. All rats except the normal control group were fasted for 24 hours (water was allowed). After anesthesia with isoflurane, 0.5 mL / rat of TNBS ethanol solution (18 mg TNBS / rat) was administered rectally via a latex tube. The tube was inserted approximately 8 cm into the rectum. After the tube was removed, the rats remained anesthetized and held in a tilted position for 15 minutes. The normal control group rats did not undergo rectal intubation stimulation. After the animals regained consciousness, they were randomly divided into groups, and drug administration began immediately (D1). The blank control group and the model group received the same amount of solvent via oral gavage once daily for 7 consecutive days (D1-D7). The animals' condition, fecal condition (presence of loose stools or bloody stools), and body weight were monitored daily. The day after the last administration (D8), the animals were deeply anesthetized with CO2, euthanized, and dissected. The entire colon was harvested, dissected along the mesenteric side, and the colonic contents were washed with physiological saline. The colon was weighed and its length was measured. The ulcer surface was measured using ImageJ software. The gross condition of the colon was observed and photographed. Tissue from the lesion site was fixed in formalin and subjected to HE staining for histopathological observation.

[0987] Result: (1) As Figure 46a As shown, the effects on the general condition of rats with TNBS-induced ulcerative colitis: rats had loose stools on the second day after TNBS modeling; the modeled rats also had symptoms of reduced appetite, fatigue, emaciation, piloerection, and dull hair. The stools of some rats improved in the later stages of drug administration, but some rats still had loose stools until the end of the experiment, and a few rats had obvious abdominal distension in the later stages of the experiment; (2) Figure 46b As shown, the effects on colon weight and length in rats with TNBS-induced colitis: Based on the combined results of colon weight per unit length (colon weight / colon length) and colon ulcer area in rats, it can be seen that the test substances (Examples 13B, 36, and 37) can all reduce colon weight per unit length and colon ulcer area in rats, with Example 13B showing a more significant effect.

[0988] Experiment 23. Decreased blood glucose in DB mice

[0989] Experimental and Analytical Methods: The leptin receptor gene (OB-R), also known as the diabetes gene (Diabetes Gene, db), is closely related to obesity, hypertension, diabetes, and lipid metabolism disorders. The course of this disease is greatly influenced by genetic background. Topical insulin cannot control the increase in blood glucose and hepatic gluconeogenesis. Leptin gene mutant mice were constructed using gene editing and embryo injection techniques. Blood glucose monitoring of homozygous mice revealed a significant difference in blood glucose levels compared to wild-type controls, making them suitable for type II diabetes research. These mice exhibited a blood glucose spike of 30 mmol / L starting at 8 weeks of age, while normal background mice had blood glucose levels of only around 6 mmol / L. We administered the drugs starting at 8 weeks of age, using small molecules from Examples 28, 29, and 30, all at 80 mpk, via gavage once daily. Twenty mice were used in each group, and blood glucose was monitored weekly from 8 weeks of age until sacrifice at 20 weeks of age.

[0990] Results: The results are as follows Figure 47 The results show that Examples 28 and 29 both have a certain blood sugar lowering effect, but Example 30 has the best blood sugar lowering effect, reaching the level of normal background mice.

[0991] Experiment 24. DIO mice showed a decrease in body weight and body fat percentage.

[0992] Experimental and Analytical Methods: The DIO model is a high-fat diet-induced obesity model. This experiment used mice of two strains, C57BL / 6J and C57BL / 6N, fed a high-fat diet containing 60% fat. Mice were switched to a high-fat diet at 7 weeks of age to establish the model. The high-fat diet was maintained for 5 months until the mice reached a weight of over 50g. Drug administration was then initiated, and weight was recorded weekly. Body fat percentage was measured every two weeks using an Echo MRI small animal body composition analyzer. We tested the weight-loss effects of five small molecules in Examples 33, 34, 35, 36, and 37, at a dose of 80mpk, administered by gavage every two days.

[0993] Result: As Figures 48a to 48d As shown, Examples 34 and 35 exhibited the best weight loss effects, reducing the weight of obese model mice from over 50g to approximately 35g, a level comparable to normal mice. Examples 33, 36, and 37 also showed weight loss effects, reducing weight to around 40g. Regarding body fat percentage, all the small molecules effectively burned fat and reduced body fat percentage in the mice; Examples 34 and 35 again demonstrated the best fat-burning effect, followed by Examples 33, 36, and 37.

[0994] Experiment 25. NOD-Scid immunodeficient mice were inoculated with Raji-Luc hematologic malignancies.

[0995] Experimental and Analytical Methods: The objective of this experiment was to test the in vivo killing effect of six small molecules on hematologic malignancies. Eighty 8-week-old female NOD-Scid immunodeficient mice were used as inoculation mice for Raji-Luc cells. In vivo fluorescence imaging was performed on day 9 post-inoculation. Based on fluorescence intensity, the 60 mice with the highest mid-range fluorescence intensity values ​​were selected for subsequent experiments. After the first in vivo imaging, drug administration began, with a dose of 100 mpk administered once daily by gavage. The small molecules tested were those from Examples 25A, 25B, 28, 38, 39, and 40, with 10 mice in each group. In vivo imaging was performed twice weekly after the start of drug administration to observe the development of hematologic malignancies in mice.

[0996] Result: As Figure 49 As shown, the control group exhibited very high fluorescence intensity, while the dosing groups showed a significant decrease in fluorescence intensity. Groups 28 and 38 were excessively toxic, with all cells dying within 21 days, resulting in no fluorescence values. Examples 25A, 25B, 39, and 40, however, demonstrated relatively good tumor-killing effects.

[0997] Experiment 26. Detection of cell proliferation using small molecule compounds

[0998] Experimental and analytical methods: Cells were seeded into capped 96-well cell culture plates. The highest concentration of the test compound was 10 μM, diluted 3-fold to a series of test concentrations. Cells were incubated at 37°C and 5% CO2 for 72 hours. The DMSO concentration was 0.5%. 100 μL of culture medium was removed from each well. 100 μL of reagent (Celltitute Glo assay kit) was added to each well, and the plate was shaken on a plate shaker (protected from light) for 2 minutes. The culture plates were incubated at room temperature (protected from light) for 30 minutes. Chemiluminescence values ​​were recorded using a multi-plate reader. The experimental results are as follows:

[0999] Table 13 Inhibition of lymphoma cell growth in vitro by compounds from the examples.

[1000]

[1001] The table shows the cell proliferation analysis of U937 (histiocytic lymphoma cells), OCI-LY3 (human diffuse large B-cell lymphoma cells), U2932 (human diffuse large B-cell lymphoma cells), and HT cells (human mixed lymphoma cells). The table shows that the compounds of this invention have an inhibitory effect on lymphoma cell proliferation. Specifically, Example 2A showed the best inhibitory activity against U937 cells, while Example 13A showed the best inhibitory effect on the proliferation of OCI-LY3 and HT cells. "—" in the table indicates no data was available.

[1002] Experiment 27. PD mouse model

[1003] Experimental and Analytical Methods: C57BL / 6 mice were used in this experiment and divided into 5 groups: a normal control group (no MPTP injection), a model group (MPTP-treated), and treatment groups for the three small molecules (Examples 13B, 25A, and 25B). Drug administration began simultaneously with modeling. Modeling lasted for 5 days, followed by intraperitoneal injection of 30 MPTP for 15 days, and then 80 MPTP for 15 days. At the endpoint, after cardiac perfusion, the brain was harvested and fixed in PFA. After complete fixation, it was transferred to sucrose for dehydration, and after complete dehydration, it was embedded in OCT, sectioned, and stained with TH. The detection index was the number of TH-positive cells in the substantia nigra. The more positive cells, the better the protective effect of the small molecules against MPTP damage.

[1004] Results: Each group consisted of 12 mice, and the statistical results are as follows: Figure 50 As shown in the T-test, **: p < 0.01, #: p < 0.05. Compared with the model group, only the Example 13B group showed statistical difference among the three small molecules, indicating that Example 13B has a good protective effect on substantia nigra neurons.

[1005] Experiment 28. COVID-19 pseudovirus infection cell model

[1006] Experimental and analytical methods: First, HEK293T cells were used to culture SARS-CoV-2 pseudoviruses that possessed only infectious ability but not replication ability. After collecting the pseudoviruses from the supernatant, normal HEK293T cells with ACE2 receptors on their surface were used as the infection targets for pseudovirus infection. After infection, the mitochondria in HEK293T cells underwent fragmentation, which could be monitored by mitochondrial fluorescent staining (mitotracker red), indicating that the cells were damaged by the pseudovirus; this was the model group. In the experimental group, cells were incubated with 300 nM of Example 13B for 1 hour before pseudovirus infection to test the protective effect of the small molecule Example 13B against viral infection.

[1007] Results: The results are as follows Figure 51The results showed that, compared with the normal control group (without pseudovirus infection), the mitochondria in the model group cells were significantly fragmented, while the mitochondria in the experimental group cells were not fragmented, indicating that the small molecule Example 13B has a good protective effect against the infection of the novel coronavirus pseudovirus.

[1008] Experiment 29. Rat model of acute traumatic brain injury (TBI)

[1009] Experimental and Analytical Methods: Male SD rats were used to establish a traumatic brain injury (TBI) rat model. Rats were randomly divided into three groups: a solvent control group (8 rats), a test substance Example 13B (80 mpk, 10 rats), and a test substance Example 26 (80 mpk, 9 rats). Drug administration began immediately after TBI surgery, administered by gavage once daily for 7 consecutive days. The endpoint (day 7) was the collection of brain tissue to detect the percentage of apoptotic cells in the hippocampus and adjacent injury areas.

[1010] Result: As Figure 52 The flow cytometry results showed that the early apoptosis rate and total apoptosis rate in the brains of rats in the test substance Example 13B (80 mpk) and test substance Example 26 (80 mpk) groups were significantly lower than those in the solvent control group (P < 0.05). Under this experimental system, repeated intravenous administration of test substances Example 13B and Example 26 significantly improved neurological function damage and brain cell apoptosis in rats with acute traumatic brain injury (TBI), and promoted the recovery of motor ability and neurological function.

[1011] Experiment 30. In vitro killing effect on pancreatic cancer cells

[1012] Experimental and Analytical Methods: We used Mia paca-2 cells as our research subject. This cell line is a pancreatic cancer cell line and is widely used in drug sensitivity testing. We performed drug sensitivity tests on 11 small molecules, with the highest concentration being 10 μM. After 3-fold dilution, we obtained a concentration gradient of 7 points. Cell viability was detected using Celltiter Glo, and dose-response curves were plotted using Graphpad Prism.

[1013] Results: The drug sensitivity curve is as follows: Figure 53 As shown, a lower IC50 value indicates greater sensitivity to the small molecule. It can be observed that Miapaca-2 cells showed the highest sensitivity to Example 26A.

[1014] Experiment 31. Inhibition of excessive urination in DB animals

[1015] Experimental and Analytical Methods: The leptin receptor gene (OB-R), also known as the diabetes gene (Diabetes Gene, db), is closely related to obesity, hypertension, diabetes, and lipid metabolism disorders. The course of this disease is greatly influenced by genetic background. Topical insulin cannot control the increase in blood glucose and hepatic gluconeogenesis. Leptin gene mutant mice were constructed using gene editing and embryo injection techniques. Blood glucose monitoring of homozygous mice of this strain revealed a significant difference in blood glucose levels compared to wild-type controls, making it suitable for type II diabetes research. These mice exhibited enormous urine output, hundreds of times that of normal mice, thus mimicking patients with frequent urination. The experiment was divided into three groups: a model group (no medication, diseased mice with high urine output), a control group (no medication, normal mice without disease), and an experimental group (administered Example 1A, 80 mpk, by gavage every two days). The control group consisted of 2 mice, and the other two groups consisted of 3 mice each. The small molecule used was Example 1A. Starting from week 8, we continuously treated the mice with the medication for 5 weeks and then observed the urine output in each cage. The larger the urine-soaked area in the cage, the greater the urine output of the mice in that cage.

[1016] Result: As Figure 54 As shown in the cage photographs, the urine-soaked area in the cages of the three-cage model group mice was the largest, while the urine-soaked area in the cages of the control group mice was almost invisible. The urine-soaked area in the cages of the drug-treated group mice was significantly reduced compared to the model group mice. This indicates that Example 1A has the function of inhibiting urinary frequency.

[1017] Experiment 32. Cardiovascular inflammation relief in APOE mutant mice fed a high-fat diet.

[1018] Experimental and Analytical Methods: Six-week-old male APOE- / - mutant B6J mice and WT B6J mice were purchased and acclimatized in the housing for two weeks. At eight weeks of age, all groups except the Vehicle and WT groups were fed a high-fat diet (Research Diets, D12492). At nine weeks of age, the Control group was given a 10% cyclodextrin solution, while the other groups were administered different small molecule compounds orally every two days (Examples 13B, 33, 36, and 37). After 16 weeks of high-fat feeding, the APOE mice were sacrificed and the aortic arch was dissected. A fixed weight of 100 mg / mouse aortic vascular epithelial tissue was analyzed using MSD (Multifactorial Determination) to determine the expression levels of various inflammatory factors, including IL-10, IL-1β, and KC / GRO.

[1019] Result: As Figures 55a to 55cAs shown, all three types of inflammatory factors showed a decreasing trend under the intervention of small molecules, with the decrease in Example 13B being the most significant, indicating that the small molecules shown have an anti-cardiovascular inflammatory effect.

[1020] Experiment 33. Example 13B: Pharmacological experiment on the effect of subthreshold hypnotic doses of sodium pentobarbital on sleep in mice.

[1021] Experimental and Analytical Methods: The effect of a single oral gavage administration (Example 13B) on the sleep latency and duration of sleep induced by a subthreshold hypnotic dose of sodium pentobarbital in ICR mice was investigated to determine whether the test substance has a hypnotic effect, and a pharmacodynamic comparison was made with the positive control drug diazepam. After acclimatization, 50 qualified animals were selected according to their body weight for the experiment and randomly divided into 5 groups using an Excel randomization method: blank control group, positive control group (diazepam), and high, medium, and low dose groups of the test substance, with 10 animals in each group. Animals in each group were given the prescribed drug solution and solvent. One hour after administration, each group of animals was intraperitoneally injected with the maximum subthreshold dose of sodium pentobarbital (a preliminary experiment was conducted to determine the maximum subthreshold hypnotic dose of sodium pentobarbital). The sleep latency, the number of animals falling asleep within 30 minutes (those whose righting reflex disappeared for more than 1 minute), and the duration of sleep for each mouse were recorded.

[1022] Result: As Figure 56a and 56b As shown, 1 mg / kg diazepam significantly shortened the sleep latency in mice. Compared with the blank control group, small molecule compounds in Examples 13B, 39, and 40 all shortened the sleep latency in mice, showing a quantitative-response relationship, but without statistical significance. Furthermore, the effects of small molecule compounds in Examples 13B, 39, and 40 in shortening the sleep latency in mice were significantly weaker than those of 1 mg / kg diazepam. Compared with the blank control group, the positive control group, administered 1 mg / kg diazepam, significantly prolonged the sleep of mice. Compared with the blank control group, small molecule compounds in Examples 13B, 39, and 40 prolonged the sleep time in mice, but without statistical significance. Furthermore, the effects of small molecule compounds in Examples 13B, 39, and 40 in prolonging the sleep time in mice were significantly weaker than those of 1 mg / kg diazepam.

[1023] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, this specification should be considered illustrative rather than restrictive.

Claims

1. The use of a derivative based on the sarcosinate-glucanogene structure in the preparation of a medicament for treating diseases associated with mitochondrial dysfunction, characterized in that, The structural formula of the derivative is as follows: The associated disease caused by the aforementioned mitochondrial dysfunction is frequent urination; or, The structural formula of the derivative is as follows: , , , The associated disease caused by the mitochondrial dysfunction is ischemic stroke.

2. The use of a derivative based on the sarcosinate-glucanogene structure in the preparation of a medicament for treating diseases associated with mitochondrial dysfunction, characterized in that, The structural formula of the derivative is as follows: The diseases associated with mitochondrial dysfunction include sleep disorders, amyotrophic lateral sclerosis (ALS), atherosclerosis, hyperlipidemia, ulcerative colitis, and traumatic brain injury.

3. The use according to claim 2, characterized in that, The associated diseases caused by the aforementioned mitochondrial dysfunction are high cholesterol and high LDL cholesterol.

Images