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

Abstract

The present invention relates to the use of a sarsasapogenin compound for preparing a medicament for treating diseases related to mitochondrial dysfunction, characterized in that the structural formula of the compound is as shown in general formula (I): [Formula 1] TIFF2025505820000258.tif65170Through in vivo and in vitro activity models, the sarsasapogenin compounds were tested and found to have excellent cytoprotective activity, especially for various mitochondrial-related indications, and to have unexpected protective activity, especially for brain neuron cells. The compounds also have excellent blood-brain permeability, and may be widely used in the treatment of many diseases caused by mitochondrial dysfunction, which is of great value. This makes up for the shortcomings of existing technologies in the application of sarsasapogenin compounds and has important scientific and commercial application value.

Description

[Technical field]

[0001] This application claims priority to Chinese patent application filed on February 18, 2022 and bearing application number 202210152310.X, and Chinese patent application filed on February 10, 2023 and bearing application number 202310093727.8, the contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of pharmaceutical technology, in particular to the field of medicinal chemical synthesis, and in particular to a novel use of derivatives based on sarsasapogenin-based structures and pharmaceutical compositions thereof for preparing therapeutic drugs for related diseases caused by mitochondrial dysfunction. [Background technology]

[0003] Timo is a common herbal medicine in China. It is the dried tuber of Anemarrhena asphodeloides Bunge, a plant of the lily family. It is a commonly used yin-nourishing agent that relieves dry mouth, relieves fever, expels evil spirits, and removes swelling in the limbs. Its extract has been proven to have biological activities such as diuretic, antidiabetic, antiplatelet aggregation, antifungal, and metabolic regulation, and has also shown inhibitory effects on cyclic AMP phosphodiesterase. The main chemical components of the extract include steroid saponins, diphenylpyrone series, polysaccharides, and lignins. Among them, steroid saponins include timosaponins AI, A-II, A-III, A-IV, BI, B-II, and B-III, marcogenin 3-O-β-D-glucopyranosyl (1→2)-β-D-glucopyranoside B, desgalactigonin, F-gitonin, and smilagenin. It also contains thymopolysaccharides A / B / C / D, cis-hinokiresinol, monomethyl-cis-hinokiresinol, oxy-cis-hinokiresinol, 2,6,4'-trihydroxy-4-methoxybenzophenone, p-hydroxyphenylcrotonic acid, pentadecanoic acid vinyl ester, β-sitosterol, mangiferin, nicotinic acid, nicotinamide and pantothenic acid.

[0004] Mitochondrial function and behavior are central to human physiology, and mitochondria perform a variety of interconnected functions, not only producing ATP and many biosyntheses, but also contributing to cellular stress responses such as autophagy and apoptosis. Mitochondria form dynamic, interconnected networks and are tightly coupled with other cellular compartments. Mitochondria also affect the physiology and organization of living organisms by functioning across cellular boundaries and regulating intercellular communication. The mitochondrial respiratory chain is mainly composed of mitochondrial respiratory chain enzymes, and defects in the mitochondrial respiratory chain enzyme complexes are an important cause of mitochondrial diseases (approximately 30-40% of mitochondrial diseases are due to defects in mitochondrial respiratory chain enzymes). Structure and function of human respiratory chain supercomplexes. Oxidative phosphorylation is carried out stepwise by five respiratory chain protein complexes present in the inner mitochondrial membrane. These five respiratory chain 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).

[0005] Therefore, it is not surprising that mitochondrial dysfunction is a cause of various diseases, including neurodegenerative and metabolic disorders. The literature also introduces 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 that target respiratory chain complexes and act on various indications are available on the market, providing multiple options for curing diseases. At present, drugs targeting respiratory chain complexes are available on the market, and their indications include tumors and cancers, cardiovascular diseases, endocrine diseases, immune diseases, coronavirus infections, inflammation, metabolic diseases, neurodegenerative diseases, intestinal diseases, autism, schizophrenia, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, and chronic fatigue syndrome.

[0006] Small molecules acting on mitochondria maintain the complex in an active state, allosterically control the activity and electron transfer efficiency of the complex, and stabilize the overall structure of the complex, thereby reducing mitochondrial ROS (superoxide free radicals) caused by electron leakage. Modulation of the complex activity can achieve global control of the energy metabolic pathway in cells and even organisms, and can also affect epigenetic modifications of the genome.

[0007] Stroke, also known as paracetamol, includes ischemic stroke (cerebral infarction) and hemorrhagic stroke (cerebral parenchymal hemorrhage, intraventricular hemorrhage, subarachnoid hemorrhage). According to the World Health Organization (WHO) definition, stroke is a multifactorial event that causes damage to cerebrovascular tissue and focal (or global) brain tissue damage, resulting in clinical symptoms for more than 24 hours or death. It is characterized by high morbidity, disability, recurrence, and mortality rates. Stroke is the leading cause of death in China.

[0008] Amyotrophic lateral sclerosis, also known as motor neuron disease, is a chronic progressive degenerative disease of upper and lower motor neurons and the muscles of the trunk, limbs, head, and face that are innervated by motor neurons. It is generally characterized by progressively worsening muscle weakness, muscle atrophy, and fasciculations caused by combined damage to upper and lower motor neurons. Clinical symptoms include progressively worsening skeletal muscle weakness, muscle atrophy, fasciculations, bulbar palsy, and pyramidal signs. In the early stages, patients may present with mild symptoms such as weakness, throbbing, and fatigue, which may be easily confused with other diseases, and gradually progress to generalized muscle atrophy, difficulty swallowing, and eventually respiratory failure. The main theories regarding the cause of motor neuron disorder include 1) accumulation of neurotoxic substances, which causes glutamate to accumulate between nerve cells and, over time, damage the nerve cells; 2) free radicals damage the nerve cell membrane; and 3) lack of nerve growth factor, which prevents nerve cells from continuing to grow and develop. Rilutek is the only drug internationally recognized and approved by the U.S. Food and Drug Administration for the treatment of amyotrophic lateral sclerosis, and should be administered as soon as possible. In addition, neurotrophic factors, antioxidants such as vitamin E and vitamin C, creatine, and CoQ10 have been tried worldwide in combination with Rilutek as protective therapies for amyotrophic lateral sclerosis, but have not yet been proven in clinical trials.

[0009] Neurodegenerative diseases are divided into acute neurodegenerative diseases and chronic neurodegenerative diseases. Acute neurodegenerative diseases mainly include stroke, brain injury (BI) and epilepsy, while chronic neurodegenerative diseases mainly include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), various types of spinocerebellar ataxia (SCA) and Pick's disease. The etiology of neurodegenerative diseases includes the following four causes: 1. Oxidative stress. Oxidative stress is caused by the excessive production and / or delayed clearance of free radicals and an imbalance between oxidative and antioxidant activity in the body, resulting in damage to cells and tissues in the body. Free radicals are atoms or groups with an unpaired electron, including hydroxyl radicals, superoxide anion, nitric oxide, etc. In recent years, oxidative damage to neural tissues has been recognized in neurodegenerative diseases such as AD, PD, and ALS. 2. Mitochondrial dysfunction. Defects in mtDNA and abnormal oxidative phosphorylation are present in the brains of AD patients. Polymerase chain reaction (PCR) and Southern blot assays revealed sporadic occurrence of mtDNA breaks, base deletions, and mistranslation mutations in brain tissues of AD patients. Electron microscopy confirmed increased mitochondrial numbers, structural abnormalities, and the appearance of lamellipodia and crystalline inclusions. In addition, when mitochondrial dysfunction occurs in neurons of AD patients, it leads to insufficient energy supply to neurons and massive ROS release, which causes oxidative stress damage, calcium regulation imbalance, and ultimately neuronal apoptosis. 3. Excitotoxicity. When the concentration of glutamate in the intercellular space is too high, toxins are produced for neurons, leading to degeneration, aging, and death of neurons. Glutamate excitotoxicity is closely related to the occurrence and progression of various neurodegenerative diseases, and is one of the important mechanisms that cause neuronal death in neurodegenerative diseases. 4. Immune inflammation. A large body of evidence has confirmed that inflammation plays an important role in the development of AD. The innate immune system is a natural immune defense function formed during the development and evolution of germ cells. Compared with other specific immune responses in the body, it can rapidly respond to various harmful substances to protect the body. Activation of the innate immune system itself can be considered a double-edged sword. Long-term and uncontrollable stimulation of harmful substances (such as aggregated Aβ) can activate the innate immune system and have harmful effects on the brain.

[0010] Corona Virus Disease 2019 (COVID-19), named "COVID-19" by the World Health Organization, is an acute respiratory infection caused by a new coronavirus infection in 2019. On March 11, 2020, the World Health Organization assessed the current COVID-19 outbreak as a global pandemic. Clinical symptoms of pneumonia patients infected with the new coronavirus are mainly fever, fatigue, and dry cough, and oxygen deficiency and hypoxia occur. Approximately half of patients develop respiratory distress after more than a week, and in severe cases, the disease progresses rapidly to acute respiratory distress syndrome, septic shock, difficult-to-correct metabolic acidosis, bleeding, and coagulation dysfunction. Currently, the main prevention methods for COVID-19 are vaccination and isolation, and treatments include paxlobid, azuvudine tablets, monoravir capsules, Sankankashu granules, and Qingfei Paidu Tang.

[0011] The search for new drugs with high efficacy, high selectivity, low toxicity, and excellent brain penetration is still extremely difficult, and there is an urgent need to prevent, control, and treat COVID-19, reduce mortality, and eliminate sequelae. Reducing free radical levels in the brain is considered a treatment, and sarsasapogenin compounds have been shown to reduce free radical levels, protect mitochondria, and use oxygen more efficiently.

[0012] Therefore, the development and rational use of compounds for the treatment of diseases caused by mitochondrial respiratory chain disorders is of great practical value. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Jodi Nunnari, et al., Mitochondria: In Sickness and in Health, Cell, Vol. 148, Issue 6, p1145-1159, March 16, 2012 Summary of the Invention [Problem to be solved by the invention]

[0014] The object of the present invention is to overcome the above-mentioned drawbacks in the prior art, and in order to achieve the above-mentioned object, the first aspect of the present invention provides the use of a derivative based on a sarsasapogenin-based structure for preparing a therapeutic drug for related diseases caused by mitochondrial dysfunction, the main feature of which is that the structural formula of the derivative is shown in general formula I. [Means for solving the problem]

[0015] [ka]

[0016] The derivative represented by the general formula I is a compound in which the following fragment A and fragment B are bonded together: [ka] where Z is NR 1 R 2 and R 1 and R 2 are each independently hydrogen, substituted or unsubstituted C-C 10 Alkyl group, C1-C 10 The substituents of the alkyl group are halogen, hydroxyl group, amino group, nitro group, cyano group, C1-C4 alkyl group, C1-C4 halogen-substituted alkyl group, C1-C4 alkoxy group, C3-C6 cycloalkyl group, C2-C4 alkenyl group, C2-C4 alkynyl group, phenyl group, benzyl group, C3-C 14 Heterocyclic groups, C3-C 14heteroaryl groups, the heteroatoms being selected from one or more of N, O, and S, or R 1 and R 2 and form a 3- to 8-membered ring, and the 3- to 8-membered ring is 10 Alkyl groups, C3-C 10 Cycloalkyl groups, C6-C 20 Aryl groups, C3-C 14 Heteroaryl groups, halogens, hydroxy groups, amino groups, alkoxy groups, -CF3, -SF5, or heteroatoms are sulfur, oxygen, NH or NR a and the heteroatom is selected from one or more of N, O, and S; X is C(O) or S(O)2; Y is C(R d )(R e ), C(O) or S(O)2, and R d , R e are each independently hydrogen or a C1-C 10 Alkyl groups, C3-C 10 Cycloalkyl groups, C6-C 20 Aryl groups, or C3-C 14 A heteroaryl group, the substituents of which are selected from halogen, hydroxy, amino, nitro, cyano, aldehyde, carboxyl, alkoxy, -CF3 or -SF5, and the heteroatoms are selected from one or more of N, O and S, or R d and R e and form a 3- to 8-membered ring, and the 3- to 8-membered ring is a C1-C10 alkyl group, a C3-C10 cycloalkyl group, or a C 6- C 20 Aryl groups, C3-C 14 Heteroaryl groups, halogens, hydroxy groups, amino groups, alkoxy groups, -CF3, -SF5, or heteroatoms are sulfur, oxygen, NH or NR a and the heteroatom is selected from one or more of N, O, and S; X2 is O, S or NH; R a are independently hydrogen or a C1-C 10 Alkyl groups, C3-C 10 Cycloalkyl groups, C6-C 20 Aryl groups, or C3-C 14 a heteroaryl group, the substituents of which are selected from halogen, hydroxy, amino, nitro, cyano, alkoxy, -CF3, or -SF5, and the heteroatoms are selected from one or more of N, O, and S; n is an integer from 0 to 10, and n is not 0, and 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, and m is 0; R3, R 4a , R 4b , R 5a , R 5b are each independently hydrogen or selected from a halogen, a substituted alkyl group, a hydroxyl group, and an amino group; [ka] represents a single or double bond, each [ka] independently indicate the racemic, S or R configuration.

[0017] Preferably, the structure of the derivative is shown in general formula (II): [ka]

[0018] Preferably, the structure of the derivative is shown in general formula III: [ka]

[0019] Preferably, the structure of the derivative is shown in general formula IV: [ka]

[0020] Preferably, the structure of the derivative is shown in general formula V: [ka] where R6 is hydrogen, substituted or unsubstituted C1-C 10 Alkyl group, C1-C 10 the substituent of the alkyl group is selected from halogen, hydroxy group, -NH2, nitro group, -CN, C1-C4 alkyl group, C1-C4 halogen-substituted alkyl group, C1-C4 alkoxy group, C3-C6 cycloalkyl group, C2-C4 alkenylene group, C2-C4 alkynyl group, phenyl group, benzyl group, pyridinyl group, -CO alkyl group, -CO aryl group, -SO2 alkyl group, -SO2 aryl group, -CO2 alkyl group, C2-C4(CO) alkenylene group, -CO2 aryl group, and -SO3H; L is hydrogen, substituted or unsubstituted C1-C 10 Alkyl group, C1-C 10 the substituent of the alkyl group is selected from halogen, a hydroxy group, -NH2, a nitro group, -CN, a C1-C4 alkyl group, a C1-C4 halogen-substituted alkyl group, a C1-C4 alkoxy group, a C3-C6 cycloalkyl group, a C2-C4 alkenyl group, and a C2-C4 alkynyl group; n is an integer from 0 to 10, n2 is 0, 1, 2, or 3; m and m' are independently an integer of 1 to 4; W1 is C or NH; V1 is C or NH; M is C, S, O or NH; Preferably, the structure of the derivative is shown in general formula VI: [ka] Y1 is C(Rd)(Re), C(O) or S(O)2, and Rd and Re are independently hydrogen or a C1-C1 having at least one substituent. 10 Alkyl groups, C3-C 10 Cycloalkyl groups, C6-C 20 Aryl groups, or C3-C 14 A heteroaryl group, the substituents of which are selected from halogen, hydroxy, amino, nitro, cyano, aldehyde, carboxyl, alkoxy, -CF3 or -SF5, and the heteroatoms are selected from one or more of N, O and S, or R d and R e and form a 3- to 8-membered ring, and the 3- to 8-membered ring is 10 Alkyl groups, C3-C 10 Cycloalkyl groups, C6-C 20 Aryl groups, C3-C 14 Heteroaryl groups, halogens, hydroxy groups, amino groups, alkoxy groups, -CF3, -SF5, or heteroatoms are sulfur, oxygen, NH or NR a and the heteroatom is selected from one or more of N, O, and S; L is hydrogen, substituted or unsubstituted C1-C 10 Alkyl group, C1-C 10 the substituent of the alkyl group is selected from halogen, a hydroxy group, -NH2, a nitro group, -CN, a C1-C4 alkyl group, a C1-C4 halogen-substituted alkyl group, a C1-C4 alkoxy group, a C3-C6 cycloalkyl group, a C2-C4 alkenyl group, and a C2-C4 alkynyl group; n is an integer from 0 to 10, n2 is 0, 1, 2, or 3; n3 is an integer from 1 to 10, m is an integer from 0 to 10; Preferably, the structure of the derivative is shown in general formula VII: [ka] where R, R are independently hydrogen, substituted or unsubstituted C-C10 Alkyl group, C1-C 10 the substituent of the alkyl group is selected from halogen, hydroxy group, -NH2, nitro group, -CN, C1-C4 alkyl group, C1-C4 halogen-substituted alkyl group, C1-C4 alkoxy group, C3-C6 cycloalkyl group, C2-C4 alkenylene group, C2-C4 alkynyl group, phenyl group, benzyl group, pyridinyl group, -CO alkyl group, -CO aryl group, -SO2 alkyl group, -SO2 aryl group, -CO2 alkyl group, C2-C4(CO) alkenylene group, -CO2 aryl group, and -SO3H; L is hydrogen, substituted or unsubstituted C1-C 10 Alkyl group, C1-C 10 the substituent of the alkyl group is selected from halogen, a hydroxy group, -NH2, a nitro group, -CN, a C1-C4 alkyl group, a C1-C4 halogen-substituted alkyl group, a C1-C4 alkoxy group, a C3-C6 cycloalkyl group, a C2-C4 alkenyl group, and a C2-C4 alkynyl group; W2 is C or NH; V2 is C, O, S or NH; n is an integer from 0 to 10, n1 is an integer from 1 to 10, n2 is 0, 1, 2, or 3.

[0021] Preferably, the structure of the derivative is shown in general formula VIII: [ka] Z1 is hydrogen, substituted or unsubstituted C1-C 10 Alkyl group, C1-C 10the substituent of the alkyl group is selected from halogen, hydroxy group, -NH2, nitro group, -CN, C1-C4 alkyl group, C1-C4 halogen-substituted alkyl group, C1-C4 alkoxy group, C3-C6 cycloalkyl group, C2-C4 alkenylene group, C2-C4 alkynyl group, phenyl group, benzyl group, pyridinyl group, -CO alkyl group, -CO aryl group, -SO2 alkyl group, -SO2 aryl group, -CO2 alkyl group, C2-C4(CO) alkenylene group, -CO2 aryl group, and -SO3H; W3 is C, S, O or NH; n is an integer from 0 to 10, n4, n5, n6, and n7 are integers from 1 to 4.

[0022] In another preferred embodiment, fragment B in the structural formula of the derivative has the following structure:

[0023] [ka]

[0024] Preferably, said derivative is either a compound, a mixture of diastereomers of the compound, or an enantiomer of the compound, [ka]

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[0060] Preferably, said derivatives include the corresponding deuterated compounds resulting from the replacement of any one or more of said hydrogen atoms by the stable isotope deuterium.

[0061] In another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of general formula I, a pharma- ceutically acceptable salt, stereoisomer, tautomer, prodrug or a pharma- ceutically acceptable carrier thereof. Preferably, the additional therapeutic agent comprises an antidepressant, an antimanic drug, an anti-Parkinson's drug, an anti-Alzheimer's drug, or a combination thereof.

[0062] Preferably, said pharma- ceutically acceptable salt is selected from any of the group: hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, triflate, benzenesulfonate, p-toluenesulfonate (toluenesulfonate), 1-naphthalenesulfonate, 2-naphthalenesulfonate, acetate, trifluoroacetate, malate, tartrate, citrate, lactate, oxalate, succinate, fumarate, maleate, benzoate, salicylate, phenylacetate, mandelate.

[0063] Preferably, the additional therapeutic agent is moclobemide, toloxatone, fluoxetine, paroxetine, citalopram, sertraline, venlafaxine, trimipramine, trazodone, imipramine, desipramine, clomipramine, amitriptyline, nortriptyline, doxepin, maprotiline, loxapine, amoxapine, mirtazapine, buspirone, chlormezanone, tandospirone, lithium carbonate, tacrine, huperzine A, galantamine, donepezil, rivastigmine, memantine, pramipexole, talipexole, ropinirole, or a combination thereof.

[0064] The invention also provides the use of a pharmaceutical composition in the preparation of a medicament for preventing, treating, curing or ameliorating a disease, illness or condition in a patient.

[0065] Preferably, the associated disease, illness or condition caused by mitochondrial dysfunction is an associated disease, illness or condition caused by respiratory chain abnormality, specifically including four types: metabolic disease, tumor, inflammation, and central nervous system disease.

[0066] Metabolic diseases include hyperglycemia, hyperlipidemia, hypercholesterolemia, high low-density lipoprotein, low high-density lipoprotein, angiogenesis diseases, non-alcoholic fatty liver disease, cerebrovascular disease, myocardial infarction, atherosclerosis, coronary artery disease, anti-aging, urinary urgency / frequency, type I diabetes, and chronic obstructive pulmonary disease.

[0067] The tumors include benign prostatic hyperplasia, Wegener's granulomatosis, pulmonary sarcoidosis, leukemia, lymphoma, pancreatic adenocarcinoma, and neuronal tumors.

[0068] Inflammation includes peripheral neuritis, chemotherapy-induced peripheral neuritis, autoimmune diseases, diseases associated with organ transplantation, influenza virus, coronavirus (prevention of infectious diseases, treatment and removal of sequelae), 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, sicca syndrome, systemic sclerosis, antiphospholipid syndrome, vasculitis, osteoarthritis, autoimmune hepatitis, autoimmune liver and gallbladder disease, primary sclerosing cholangitis, nephritis, gluten-sensitive enteropathy, autoimmune thrombocytopenia, transplant rejection, ischemia-reperfusion injury of solid organs, sepsis, periodontitis, systemic inflammatory response syndrome, myocarditis, allergic diseases, asthma, interleukin-I converting enzyme-associated fever syndrome, Behcet's disease, etc.

[0069] Central nervous system diseases include Pick's disease, spinal cord injury repair, depression, anxiety, Parkinson's disease, Alzheimer's disease, sleep disorders, 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, muscle weakness, etc. Effect of the Invention

[0070] Using the compounds, pharmaceutical compositions and their uses of the present invention based on sarsasapogenin system, the sarsasapogenin system compounds were tested through in vivo and in vitro activity models, and it was unexpectedly found that many derived compounds have excellent cytoprotective activity, especially unexpected protective activity on brain neuron cells, and the compounds have very good blood-brain permeability, which may be widely used in the treatment of many diseases caused by mitochondrial dysfunction, and are of great value, which makes up for the shortcomings of existing technologies in the application of sarsasapogenin system compounds and has important scientific and commercial application value. [Brief description of the drawings]

[0071] [Figure 1] FIG. 1 shows the neuronal cell death profile under the action of various small molecule compounds. [Diagram 2] FIG. 2 shows hydrogen peroxide (H2O2)-induced oxidative damage by small molecule compounds to human SHSY5Y neural tumor cells. [Diagram 3] FIG. 3 shows the proportion of neuronal dead cells. [Figure 4] Figure 4 shows the phenotype of the effect of each experimental group on inflammation in zebrafish. [Diagram 5] FIG. 5 shows the effect on zebrafish inflammation (neutrophil counts). [Figure 6] FIG. 6 shows the anti-inflammatory effects of each experimental group on inflamed zebrafish. [Figure 7] FIG. 7 shows changes in cerebral blood flow during surgery in mice. [Figure 8] FIG. 8 shows changes in mouse body weight before and after surgery. [Figure 9] FIG. 9 shows changes in forelimb grip strength in mice. [Figure 10] FIG. 10 shows the neurological deficit scores of the mice. [Figure 11] FIG. 11 shows the cerebral infarction volume in mice. [Figure 12] FIG. 12 shows the brain edema volume in mice. [Figure 13] FIG. 13 shows immobility time in the mouse FST. [Figure 14] FIG. 14 shows the sugar-water preference rate in SPT mice. [Figure 15] FIG. 15 shows immobility time in mouse TST. [Figure 16] FIG. 16 shows the measurement of ROS levels in mouse serum. [Figure 17] FIG. 17 shows the measurement of H2O2 concentration in mouse serum. [Figure 18] FIG. 18 shows the NO concentration measurement in mouse serum. [Figure 19] FIG. 19 shows the measurement of lipid peroxidation levels in mouse serum. [Figure 20] FIG. 20 shows ROS level measurements in mouse hippocampus. [Figure 21] FIG. 21 shows the measurement of H2O2 concentration in the mouse hippocampus. [Figure 22] FIG. 22 shows NO concentration measurements in mouse hippocampus. [Diagram 23] FIG. 23 shows the measurement of lipid peroxidation levels in mouse hippocampus. [Figure 24] FIG. 24 shows the measurement of IL-1β concentration in mouse serum. [Diagram 25] FIG. 25 shows the measurement of IL-6 concentration in mouse serum. [Figure 26] FIG. 26 shows the measurement of IL-10 concentration in mouse serum. [Figure 27] FIG. 27 shows the measurement of IL-1β concentration in the mouse hippocampus. [Figure 28] FIG. 28 shows the measurement of IL-6 concentration in the mouse hippocampus. [Figure 29] FIG. 29 shows the measurement of IL-10 concentration in mouse serum. [Diagram 30] 30a to 30b show the binding strength between each small molecule and respiratory chain complex I, respectively. [Diagram 31] 31a to 31b show the binding strength between each small molecule and respiratory chain complex I, respectively. [Diagram 32]32a to 32b show the binding strength between each small molecule and respiratory chain complex I, respectively. [Diagram 33] FIG. 33 shows the activity results of each small molecule against SMP. [Diagram 34] FIG. 34 is a schematic diagram showing the effect of each small molecule on cellular oxygen consumption. [Diagram 35] 35a-35b show the effects of small molecules on mitochondrial ROS and mitochondrial transmembrane potential, respectively. [Fig. 36a-b] 36a-36b show the results of small molecules on atherosclerosis in APOE mice, respectively. [Fig. 36c-d] 36c-36d show the results of small molecules on atherosclerosis in APOE mice, respectively. [Figure 37] 37a-37g show the results of small molecules on the water maze in AD rats. [Figure 38] FIG. 38 shows the results of small molecules on the T-maze in AD rats. [Figure 39] 39a-39b show the results of small molecules on nest-building behavior in AD mice. [Diagram 40] 40a and 40b show the results of small molecules on the water maze in AD mice. [Diagram 41] FIG. 41 shows the results of small molecules versus light and dark boxes in AD mice. [Diagram 42] Figures 42a and 42b show the results of small molecules on survival of TDP43A315T mice. [Diagram 43] FIG. 43 shows the results of small molecules on gait analysis in SODG93A mice. [Diagram 44] FIG. 44 shows the results of small molecules on the open field test in SODG93A mice. [Diagram 45] 45a and 45b show the results of small molecules on the DSS mouse colitis model. [Figure 46] 46a and 46b show the results of small molecules on the TNBS rat colitis model. [Figure 47] FIG. 47 shows the results of small molecules on blood glucose in DB mice. [Figure 48] 48a to 48d show the results of the body weight and body fat percentage of DIO mice. [Figure 49] FIG. 49 shows the in vivo killing effect of small molecules on hematomas. [Figure 50] FIG. 50 shows the results of the protective effects of small molecules on substantia nigra neurons. [Figure 51] FIG. 51 shows the results of the protective effect of small molecules against viral entry. [Figure 52] FIG. 52 shows the results of small molecules on a rat TBI acute traumatic brain injury model. [Figure 53] FIG. 53 shows the results of in vitro killing effects of small molecules on pancreatic adenocarcinoma cells. [Figure 54] FIG. 54 shows the effect of small molecules on excessive urination in DB animals. [Figure 55] 55a-55c show the results of small molecules on cardiovascular inflammation in high fat fed APOE mutant mice. [Figure 56] 56a and 56b show the results of pharmacodynamic experiments of small molecules on subthreshold hypnotic doses of sodium pentobarbital affecting sleep in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] In order to make the technical content of the present invention more clear, the specific embodiments of the present invention are further described below.

[0073] In the present invention, the term "alkyl group" refers to a monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, including straight-chain and branched-chain hydrocarbon groups, such as, for example, methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), s-butyl ((CH3)(CH3CH2)CH-), tert-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0074] For the purposes of the present invention, the term "alkyl group" includes substituted and unsubstituted alkyl groups.

[0075] In the present invention, the term "substituted or unsubstituted" means that the group is unsubstituted, or the H in the group can be substituted with one or more (preferably 1 to 6, more preferably 1 to 3) substituents.

[0076] In the present invention, the term "substituted" means that the group has one or more (preferably 1 to 6, more preferably 1 to 3) substituents selected from the following group: halogen, hydroxy group, -NH2, nitro group, -CN, C1-C4 alkyl group, C1-C4 halogen-substituted alkyl group, C1-C4 alkoxy group, C3-C6 cycloalkyl group, C2-C4 alkenyl group, C2-C4 alkynyl group, phenyl group, benzyl group, C2-C8 heterocyclic group, C2-C8 heteroaryl group, wherein the heteroatom is any one or more selected from N, O and S.

[0077] In the present invention, the term "cycloalkyl group" refers to a substituted or unsubstituted C-C 12 It refers to a cycloalkyl group.

[0078] In the present invention, the term "alkoxy group" refers to an -O-alkyl group, where the alkyl group can be saturated or unsaturated, branched, linear or cyclic. Preferably, the alkoxy group has 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms. Representative examples include, but are not limited to, methoxy, ethoxy, and proxoxy groups.

[0079] In the present invention, the term "aryl group" refers to a monovalent aromatic carbocyclic group having 6 to 20 (preferably 6 to 14) carbon atoms, either monocyclic (such as phenyl group) or polycyclic (such as naphthalene or anthracene group), and where the point of attachment is on an aromatic carbon, the fused ring may be non-aromatic (e.g., 2-benzoxazolone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, etc.). Preferably, aryl groups include phenyl and naphthalene groups. The term includes substituted or unsubstituted forms, the substituents being as defined above.

[0080] In the present invention, the term "alkenyl group" refers to an alkenyl group having 2 to 10 (e.g., 2 to 6 or 2 to 4) carbon atoms and at least one (e.g., 1 to 2) unsaturated alkenyl bond (>C=C<). These groups include vinyl groups, allyl groups, butyl-3-alkenyl groups, and the like.

[0081] In the present invention, the term "cycloalkyl group" refers to cyclic alkyl groups of 3 to 10 carbon atoms and monocyclic or polycyclic (including fused, bridged, and spirocyclic systems). In fused ring systems, one or more of the rings can be a cycloalkyl group, a heterocyclic aryl group, or a heteroaryl group, provided that the attachment site penetrates the ring of the cycloalkyl group. Suitable cycloalkyl groups include, for example, adamantane, cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl groups.

[0082] As used herein, the term "halogen substituted" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0083] In the present invention, the term "heteroaryl group" refers to an aryl group having 1-10 carbon atoms and 1-4 heteroatoms selected from oxygen, nitrogen and sulfur in the ring, which may be single ring (such as pyridinyl or benzothienyl) or fused ring (such as indolizinyl or benzothienyl), where the fused ring may be non-aromatic and / or contain one heteroatom, so long as the point of attachment is an atom penetrating the aromatic heteroaryl group. In certain embodiments, the nitrogen and / or sulfur ring atoms of the heteroaryl group are optionally oxidized to N-oxide (NO), sulfinyl or sulfonyl. Preferably, heteroaryl groups include pyridinyl, pyrrolyl, indolyl, thienyl and furyl groups. The term includes substituted or unsubstituted heteroaryl groups.

[0084] In the present invention, the term "substituted heteroaryl group" refers to a heteroaryl group substituted with 1 to 5, preferably 1 to 3, and more preferably 1 to 2, substituents, which are the same as those in a substituted aryl group.

[0085] As used herein, the terms "heterocycle" or "heterocyclic" or "heterocycloalkyl group" or "heterocyclic group" refer to saturated, partially saturated or unsaturated (but not aromatic) groups having single or fused rings (including bridged and spiro ring systems) and having 1-10 carbon atoms and 1-4 heteroatoms selected from oxygen, nitrogen and sulfur within the ring, where in fused ring systems one or more of the rings can be a cycloalkyl group, aryl group or heteroaryl group, so long as the point of attachment is through an aromatic heteroaryl group. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide the N-oxide, sulfinyl or sulfonyl moieties.

[0086] In the present invention, the term "substituted heterocyclic" or "substituted heterocycloalkyl group" or "substituted heterocyclic group" refers to a heterocyclic group substituted with 1 to 5 (e.g. 1 to 3) substituents, said substituents being the same as those defined for the substituted cycloalkyl group.

[0087] As used herein, the term "stereoisomer" refers to compounds that differ chirally at one or more stereocenters. Stereoisomers include enantiomers and diastereomers.

[0088] In the present invention, the term "tautomer" refers to alternative forms of a compound with different proton positions, such as enol-ketone and imine-enamine tautomers, or heteroaryl tautomers, where the heteroaryl group contains a ring atom attached to the -NH- portion of the ring and the N- portion of the ring, such as pyrazole, imidazole, benzimidazole, triazole, and tetrazole.

[0089] The present invention provides pharmaceutical compositions comprising an active ingredient within a safe and effective amount and a pharma- ceutically acceptable carrier.

[0090] In the present invention, the term "active ingredient" refers to a compound of general formula (I) described in the present invention or a pharma- ceutically acceptable salt thereof, a stereoisomer thereof or a tautomer thereof, or a prodrug thereof.

[0091] The "active ingredients" and pharmaceutical compositions described in the present invention can be used as mitochondrial protection agents. In another preferred embodiment, they are used in the preparation of a medicament for preventing and / or treating a neurodegenerative disease. In another preferred embodiment, they are used in the preparation of a medicament for preventing and / or treating a mitochondrial-related metabolic disease.

[0092] The term "safe and effective amount" refers to an amount of active ingredient sufficient to clearly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000 mg of active ingredient / agent, preferably 10-200 mg of active ingredient / agent. Preferably, the "one agent" is one tablet.

[0093] The term "pharmaceutical acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that must be of sufficient purity and sufficiently low toxicity to be applicable to humans. The term "compatible" refers to the ability of each component of the composition to be mixed with the active ingredient of the present invention and with each other without significantly decreasing the effectiveness of the active ingredient.

[0094] The compounds of the preferred embodiments of the present invention can be administered as the sole active pharmaceutical agent or in combination with one or more other agents used in the treatment of cancer.

[0095] Generally, the compound of the preferred embodiment is administered in a therapeutically effective amount in any acceptable manner through a similarly effective drug.The actual dose of the compound of the preferred embodiment (i.e., active ingredient) is determined by various factors, such as the severity of the disease to be treated, the age and relative health of the patient, the potency of the compound administered, the route and form of administration, and other factors.The drug can be administered multiple times a day, and is preferably administered once or twice a day.All these factors are within the consideration of the attending physician.

[0096] For purposes of the preferred embodiments, a therapeutically effective dose may be a total daily dose administered to a patient, typically in a single or divided dose, for example, about 0.001 to about 1000 mg / kg body weight per day, preferably about 1.0 to about 30 mg / kg body weight per day. The dosage unit composition may include a dosing factor to form a daily dose. The choice of dosage form depends on various factors, such as the mode of administration and the bioavailability of the drug substance. Generally, the compounds of the preferred embodiments, as pharmaceutical compositions, may be administered by any route suitable for treating a medical condition. Suitable routes include, but are not limited to, oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intradermal), intravaginal, intraperitoneal, intrapulmonary, and intranasal. It is understood that the preferred route of administration will vary depending on the medical condition of the patient. The preferred mode of administration is oral administration, which allows convenient adjustment of the daily dosage depending on the degree of bitterness. The compound may be formulated with a pharma- ceutically acceptable carrier or excipient, such as tablet, pill, capsule, semisolid, powder, sustained release formulation, solution, suspension, elixir, aerosol or any other suitable composition. When the compound is formulated for parenteral administration, it may be formulated with a pharma- ceutically acceptable parenteral carrier. Another preferred mode of administration of the compound of the preferred embodiment is inhalation, which is an efficient method for delivering therapeutic agents directly to the airways (see, for example, U.S. Patent No. 5,607,915).

[0097] The present invention can administer the compound in any convenient formulation form, and the term "formulation" in the present invention refers to a dosage form containing the compound of general formula I of the present invention, which is advantageous for drug delivery, including, but not limited to, aqueous solution injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder mists, sustained release agents, controlled release agents, etc. These pharmaceutical additives may be those commonly used in various formulations, such as isotonicity agents, buffers, flavoring agents, excipients, fillers, adhesives, disintegrants, lubricants, etc., or may be those selected to suit the above substances, such as emulsifiers, solubilizers, bacteriostatic agents, analgesics, antioxidants, etc. These additives can effectively improve the stability and solubility of the compound contained in the composition, or change the release rate and absorption rate of the compound, thereby promoting the metabolism of the compound of the present invention in vivo and enhancing the drug administration effect. Additionally, it may be an additive, including but not limited to, gelatin, albumin, chitosan, polyethylene glycol, polyurethane, polycarbonate, polyester polymer materials, etc., used to achieve a particular drug administration purpose or method, such as sustained release drug administration, controlled release drug administration, pulsatile drug administration, etc., such as, but not limited to, polyethylene glycol, polyurethane, polycarbonate, and copolymers thereof. Primary expressions of the term "advantageous" include, but are not limited to, improved therapeutic efficacy, improved bioavailability, reduced toxic side effects, improved patient compliance, etc.

[0098] Suitable pharma- ceutically acceptable carriers or excipients include handling agents and drug transport modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, carmellose sodium, glucose, hydroxypropyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin sodium, povidone, low melting waxes, ion exchange resins, and the like, and combinations of any two or more thereof. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol, and various oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and ethylene glycol. Other suitable pharma- ceutically acceptable excipients are described in Remington's Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991), incorporated herein by reference.

[0099] In the present invention, the term "pharmaceutically acceptable salts" refers to non-toxic acid or basic earth metal salts of compounds of general formula I. These salts can be prepared by in situ methods during the final isolation and purification of compounds of general formula I, or by reacting suitable organic or inorganic acids or bases with basic or acidic functional groups, respectively. Representative salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecyl sulfate, ethanesulfonate, glucose enanthate, glycerophosphate, hemisulfate, heptanoate, caproate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthylsulfonate, oxalate, pamoate, pectinate, thiocyanate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, thiocyanate, salicylate, p-toluenesulfonate, and undecanoate salts. In addition, nitrogen-containing basic groups can be quaternized with reagents such as alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl, and dipentyl sulfates; long chain halides, such as decyl, lauryl, myristyl, and stearalkyl chlorides, bromides, and iodides; and aryl halides, such as benzyl and phenylethyl bromides. This results in water-soluble, oil-soluble, or dispersible products. 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.The base addition salts can be prepared by in situ methods during the final isolation and purification of the compounds of general formula I, or by reacting the carboxylic acid moiety with a suitable base (e.g., pharma- ceutically acceptable hydroxides, carbonates, bicarbonates of metal cations), or ammonia, or organic primary, secondary, or tertiary amines, respectively. Pharmaceutically acceptable salts include, but are not limited to, basic metal and basic earth metal based cations such as salts of sodium, lithium, potassium, calcium, magnesium, aluminum, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Other representative organic amines used to form base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc.

[0100] In the present invention, the term "pharmaceutical acceptable prodrug" refers to a compound that is rapidly converted in vivo to the parent compound of the above general formula, for example by hydrolysis in blood, and is a prodrug of the compound of the preferred embodiment. T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of ACS 15 Symposium Series, and Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Publishing Company, 1987, provide a thorough discussion and are hereby incorporated by reference.

[0101] The present invention provides a method for preparing the compound of general formula (I). Taking sarsasapogenin as an example, the preparation method of the key intermediate is as follows:

[0102] α configuration: [ka]

[0103] β configuration: [ka]

[0104] The preparation of the compound of general formula (I) is as follows: Taking sarsasapogenin parent α configuration as an example (other configurations or other parent configurations, preparation methods are all consistent with the provided methods), the synthesis route is as follows: [ka] In each formula, R 1 , R 2 The definitions of , Y, and n are as above.

[0105] The following will further describe the present invention in accordance with specific embodiments, which should be understood to be used only to describe the present invention and not to limit the scope of the present invention.

[0106] The following abbreviations have the meanings indicated below.

[0107] DBU refers to 1,8-diazabicyclo[5.4.0]undec-7-ene, DIBAL refers to diisobutylaluminum hydride, DIAD refers to diisopropyl azodiformate, 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 dimethylsulfoxide, 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 refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, HATU refers to 2-(7-aza-1H-benzotriazo-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, HMPA refers to hexamethylphosphoramide, IPA refers to isopropyl alcohol, LDA refers to lithium diisopropylamide, LHMDS refers to lithium bis(diphenylphosphine)amide, , LAH refers to lithium aluminum hydride, PyBOP refers to (benzotriazol-1-yloxy)tripyrrolidinophosphonium 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-propylphosphate cyclic anhydride, PMA refers to phosphomolybdic acid, PE refers to petroleum ether, EA refers to ethyl acetate, RBF refers to round bottom flask, and rt refers to room temperature.

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as known to those skilled in the art. Also, any methods and materials similar or equivalent to those described may be applied to the method of the present invention. The preferred embodiments and materials described in this application are for illustrative purposes. EXAMPLES

[0109] Example 1 [ka] The specific preparation procedure is as follows:

[0110] Step 1: Intermediate 1 [ka] In a 500 mL round bottom flask, add 10 g of raw material A, dissolve in DCM (100 mL), add Dess-Martin (12.8 g) at 0°C, heat to rt after 15 min, stir and react for 1 h, and monitor the completion of the reaction (PMA coloring) by TLC. After the reaction is complete, remove the filter residue by suction filtration, spin-dry the filtrate, redissolve in DCM, wash twice with water, and obtain 9 g of white solid product (Intermediate 1) by silica gel column chromatography.

[0111] Step 2: Intermediate 2 [ka] In a 100mL round bottom flask, 5g of intermediate 1 and 1.7g of NH2OH·HCl were added, dissolved in 50mL of dry pyridine, stirred at 70℃ for 1-2h, and monitored for completion of the reaction (PMA coloring) by TLC. After completion of the reaction, the solvent was removed by spinning, redissolved in DCM, washed twice with 1N HCl aqueous solution, dried, and purified by silica gel column chromatography to obtain 5.2g of a light yellow solid crude product.

[0112] Step 3: Intermediate 3 [ka] Add 5 g of intermediate 2 to a 500 mL round bottom flask, dissolve in 100 mL of dry MeOH, add 4.2 g of NiCl2·6H2O at 0°C, add 2.7 g of NaBH4 in portions after 15 min, heat to rt after 30 min, stir and react for 4 h, and monitor the completion of the reaction (PMA coloring) by TLC. After the reaction is complete, remove the filter residue by suction filtration, spin dry the filtrate, and obtain 5 g of white solid product.

[0113] Step 4: Example 1 [ka] In a round-bottom flask, 0.055g of intermediate 3, 0.041g of 3-(4-methylpiperazin-1-yl)-propionic acid, 0.046g of EDC, 0.084 mL of triethylamine, and 0.003g of DMAP were dissolved in 2 mL of dichloromethane, and reacted at room temperature for 2 h. After the reaction was completed, the mixture was washed twice with saturated aqueous NH4Cl, dried, and purified by silica gel column chromatography to obtain 25 mg of the product. NMR data: 1 HNMR (400 MHz, 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 spectrum: [M+1]570.5.

[0114] Example 1A [ka] The specific preparation procedure of Example 1A is as follows:

[0115] Step 1: Intermediate 4 [ka] Under argon gas protection, 0.5g of sarsasapogenin raw material A, 0.4g of 4-nitrobenzoic acid, and 0.63g of PPh3 were dissolved in 5 mL of dry THF and stirred in an ice-water bath for 5 min. DIAD was then slowly added dropwise and stirred for 10 min. The ice bath was then removed and the reaction was allowed to proceed at room temperature for 3 h. The reaction was then detected. After the reaction was complete, the solvent was spin-dried, extracted with sodium bicarbonate solution / dichloromethane, purified by silica gel column chromatography PE:EA=45:1, detected by thin layer chromatography (PE:EA=15:1), and colored with vanillin. The yield was 55%.

[0116] Step 2: Intermediate 5 [ka] 0.36g of intermediate 4 and 0.35g of K2CO3 were added to 15mL of MeOH and stirred at 55℃ overnight. The solvent was spun dry, extracted with water / dichloromethane, and the organic layer was spun dry to obtain the product, which was directly put into the next reaction and developed with vanillin. The yield was 80%.

[0117] Step 3: Intermediate 6 [ka] Under argon gas protection, 0.2g of intermediate 5, 0.14g of phthalimide, and 0.25g of PPh3 were dissolved in 4 mL of dry THF and stirred in an ice-water bath for 5 min. 0.19g of DIAD was slowly added dropwise and 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 detected. The solvent was spin-dried, extracted with water / dichloromethane, purified by silica gel column chromatography PE:EA=45:1, and detected by thin layer chromatography PE:EA=15:1. The color was developed with vanillin, and the yield was 50%.

[0118] Step 4: Intermediate 7 [ka] 0.13 g of intermediate 6 and 0.072 g of N2H4·H2O were added to 10 mL of MeOH and stirred at 55 °C overnight. The solvent was spun dry, washed with water, extracted with dichloromethane, and the organic layer was spun dry to give the product, which was directly used in the next reaction and developed with vanillin. The yield was 90%.

[0119] Step 5: Example 1A [ka] Under argon gas protection, 0.07g of the intermediate, 0.058g of 3-(4-methylpiperazin-1-yl)-propionic acid, 0.065g of EDC and 0.01g of DMAP were dissolved in 4 mL of dichloromethane and reacted at room temperature for 4 hours to detect the reaction. The reaction was extracted with sodium bicarbonate solution / dichloromethane and passed through a basic alumina column PE:EA=1:1. The product was colored with vanillin and the yield was 65%. NMR data: 1 H NMR (400 MHz, CDCl3) δ 8.85 (d, J = 6.9 Hz, 1H), 4.41 (dd, J = 13.0, 6.6 Hz, 1H), 4.18 (s, 1H),3.95 (d, J = 10.6 Hz, 1H), 3.30 (d, J = 11.6 Hz, 1H), 3.01-2.20(brs, 8H), 2.62 (d, J = 3.0 Hz, 2H), 2.39(s, 2H), 2.30 (s, 3H), 1.23-2.07 (m, 27H), 1.08 (d, J = 6.7 Hz, 3H), 1.05 - 0.93 (m, 6H), 0.76 (s, 3H). Mass spectrum: [M+1] 570.5.

[0120] Example 1B [ka] The specific preparation procedure of Example 1B is as follows:

[0121] Step 1: Intermediate 8 [ka] Using the raw material A as the starting material, intermediate 8 was obtained under the same conditions as in step 3 of Example 1A in a yield of 50%.

[0122] Step 2: Intermediate 9 [ka] In this procedure, intermediate 8 was used as the raw material under the same conditions as in procedure 4 in Example 1A to give intermediate 9 in a yield of 90%.

[0123] Step 3: Example 1B [ka] In this procedure, intermediate 9 and 3-(4-methylpiperazin-1-yl)-propionic acid were used as raw materials, and the conditions were the same as in procedure 5 of example 1A to obtain example 1B in 65% yield. NMR data: 1 H NMR (400 MHz, CDCl3) δ 8.15 (d, J = 7.6 Hz, 1H), 4.33 (dd, J = 14.8, 7.5 Hz, 1H), 3.88 (dd, J =10.9, 2.4 Hz, 1H), 3.65 (m, 1H), 3.23 (d, J = 10.9 Hz, 1H), 2.90-2.10(brs, 8H), 2.56 (t, J = 6.2 Hz, 2H),2.26 (m, 2H), 2.24 (s, 3H), 1.23-2.07 (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.

[0124] Example 2 [ka] Example 2 was obtained using Intermediate 3 and 1-methylpiperidine-4-carboxylic acid under the same experimental conditions as described in Step 4 of Example 1. NMR data:1 HNMR(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 spectrum: [M+1]541.5.

[0125] Example 3 [ka] Using intermediate 3 and N,N-dimethylglycine as starting materials, Example 3 was obtained under the same experimental conditions as those described in Step 4 of Example 1. NMR data: 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 spectrum: [M+1]501.3.

[0126] Example 4 [ka] Using intermediate 3 and N,N-dimethyl-β-alanine as starting materials, Example 4 was obtained under the same experimental conditions as described in Step 4 of Example 1. NMR data: 1 HNMR(CDCl3, 400MHz): δ 0.76 (s, 3H), 0.80-2.20 (m, 36H), 2.32 (s, 3H), 2.36 (s, 3H), 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.

[0127] Example 4A [ka] Using intermediate 7 and N,N-dimethyl-β-alanine as starting materials, Example 4A was obtained under the same experimental conditions as described in Step 5 of Example 1A. NMR data: 1 H NMR (400 MHz, CDCl3) δ 9.20 (d, J = 7.8 Hz, 1H), 4.41 (dd, J = 14.1, 7.6 Hz, 1H), 4.19 (d, J = 7.2 Hz, 1H), 3.95 (dd, J = 11.0, 2.5 Hz, 1H), 3.30 (d, J = 10.9 Hz, 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 = 7.1 Hz, 3H), 1.02 - 0.92 (m, 6H), 0.76 (s, 3H). Mass spectrum: [M+1] 515.3.

[0128] Example 4B [ka] Using intermediate 9 and N,N-dimethyl-β-alanine as starting materials, Example 4B was obtained under the same experimental conditions as described in Step 5 of Example 1A. NMR data: 1 H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 7.2 Hz, 1H), 4.41 (dd, J = 14.1, 7.5 Hz, 1H), 3.95 (dd, J = 10.9, 2.3 Hz, 1H), 3.78 - 3.65 (m, 1H), 3.30 (d, J = 11.0 Hz, 1H), 2.53 (t, J = 6.2 Hz, 2H), 2.32 (t, J = 6.2 Hz, 2H), 2.27 (s, 6H), 1.23-2.07 (m, 27H), 1.08 (d, J = 7.1 Hz, 3H), 1.02 - 0.93 (m, 6H), 0.76 (s, 3H). Mass spectrum: [M+1] 515.3.

[0129] Example 5 [ka] Example 5 was obtained using intermediate 3 and 3-oxo-3-(4-methylpiperazin-1-yl)propionic acid under the same experimental conditions as described in step 4 of Example 1. NMR data: 1 HNMR(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 spectrum: [M+1]584.3.

[0130] Example 6 [ka] Example 6 was obtained using intermediate 3 and 3-oxo-3-(1-methylpiperazine-4-amino)propionic acid under the same experimental conditions as those described in step 4 of Example 1. NMR data: 1 HNMR(CDCl3, 400MHz): 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 spectrum: [M+1]598.3.

[0131] Example 7 [ka] Example 7 was obtained using intermediate 3 and 3-oxo-3-(1-methylpiperidine-4-methylamino)propionic acid under the same experimental conditions as described in step 4 of Example 1. NMR data: 1HNMR(CDCl3, 400MHz): 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 spectrum: [M+1]612.3.

[0132] Example 8 [ka] Example 8 was obtained using intermediate 3 and 3-oxo-3-(3-morpholinopropyl)aminopropionic acid under the same experimental conditions as those described in step 4 of Example 1. NMR data: 1HNMR (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 spectrum: [M+1]628.3.

[0133] Example 9 [ka] Example 9 was obtained using intermediate 3 and 3-oxo-3-(1,4-bipiperidine-1-)propionic acid under the same experimental conditions as described in step 4 of Example 1. NMR data: 1HNMR (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 spectrum: [M+1]652.3.

[0134] Example 10 [ka] Example 10 was obtained using intermediate 3 and 3-oxo-3-(piperazin-1-yl)propionic acid under the same experimental conditions as described in step 4 of Example 1. NMR data: 1H NMR (CDCl3, 400 MHz): 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 spectrum: [M+1] 570.3.

[0135] Example 11 [ka] Using intermediate 3 and 3-(hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanoic acid as starting materials, Example 11 was obtained under the same experimental conditions as those described in Step 4 of Example 1. NMR data: 1HNMR (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 spectrum: [M+1]596.4.

[0136] Example 12 [ka] 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 experimental conditions as those described in Step 4 of Example 1. NMR data: 1HNMR (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 spectrum: [M+1]667.5.

[0137] Example 13 [ka] The specific preparation procedure of Example 13 is as follows:

[0138] Step 1: Intermediate 10 [ka] In a 500 mL round bottom flask, intermediate 12 (5 g, 1 eq), mono-tert-butyl malonate (2 eq), EDC·HCl (2 eq), DMAP (0.1 eq) and Et3N (5 eq) were added, dissolved in 100 mL of dry DCM, stirred at room temperature for 2 h, and monitored for completion (PMA coloration) by TLC. After completion of the reaction, the mixture was washed twice with saturated aqueous NH4Cl, dried, and purified by silica gel column chromatography to obtain 4.5 g of product.

[0139] Step 2: Intermediate 11 [ka] Add intermediate 10 (4.5g, 1eq) to a 500mL round bottom flask and dissolve in 100mL dry DCM. Add TFA (10mL, 15eq) dropwise at 0°C, and after 15min, heat to room temperature and react for 1-2h. Monitor reaction completion (PMA coloration) by TLC. After reaction completion, spin dry, redissolve in DCM, wash twice with saturated NaHCO3 aqueous solution, dry, and subject to silica gel column chromatography to obtain 4g of white solid product, i.e. intermediate 11.

[0140] Step 3: Example 13 [ka] In a 25 mL round bottom flask, intermediate 11 (50 mg, 1 eq), intermediate 12 (2 eq), HATU (2 eq) and Et3N (5 eq) were added, dissolved in 3 mL of dry DCM, stirred at room temperature, reacted for 1 h, and monitored for completion (PMA coloring) by TLC. After completion of the reaction, the mixture was washed twice with saturated aqueous NH4Cl, dried, and purified by thin layer chromatography (dichloromethane:methanol = 10:1) to obtain 8 mg of a white solid. NMR data: 1HNMR(CDCl3, 400MHz, ppm): δ 0.76 (s, 3H), δ 0.80-2.30 (m, 36H), δ 2.55-3.00 (m, 9H), δ 3.15-4.50 (m, 14H), δ 5.25 (br s, 1H); Mass spectrum: [M+1]641.5.

[0141] Example 13A Step 1: Intermediate 13 [ka] 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, and the reaction solution was allowed to naturally warm to room temperature (23-28 ° C.). After stirring for 4 h, a pyridine (200 mL) solution of 105 g of TsCl (0.55 mol, 0.5 eq) was added to the reaction solution under N2 protection at room temperature, and after the dropwise addition was completed, the reaction was allowed to proceed overnight at room temperature (23-28 ° C.), and the system turned wine red, and the mixture was stirred at room temperature (23-28 ° C.) for 48 h. After the reaction was completed, the reaction solution was slowly added dropwise to water (65 L) at 0-10°C, and a large amount of solid precipitated. The solid was filtered and the filter cake was washed with 500 mL of PE:EA (50:1) mixed solvent. The solid was dried under vacuum (40-50°C water bath) for 8 h to obtain 451 g of solid, i.e., intermediate 13.

[0142] Step 2: Intermediate 14 [ka] A 500 mL flask was charged with intermediate 13 (25 g, 43.8 mmol), potassium acetate (8.60 g, 87.6 mmol), 18-crown-6 (23.15 g, 87.6 mmol) and 300 mL dimethyl sulfoxide solvent. The reaction was heated to 55 °C and reacted at this temperature for 16 h. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 15:1) until complete reaction. The reaction mixture was then poured into 1 L of ice water and stirred for 30 min. Filtration and washing the filter cake with water gave 7 g of a white solid.

[0143] Step 3: Intermediate 5 [ka] In a 250 mL flask, intermediate 14 (2.0 g, 4.36 mmol), 60 mL of tetrahydrofuran, 60 mL of methanol, 30 mL of water, and 5.5 mL of 4N LiOH aqueous solution were added. The reaction was heated to 60°C and allowed to react at this temperature for 2 h. The reaction was monitored by TLC and the developing system was petroleum ether:ethyl acetate=7:1. After the reaction was complete, most of the organic solvent was removed by spin filtration and 50 mL of water was added. Filtration and washing of the filter cake with water gave 1.7 g of a white solid.

[0144] Step 4: Intermediate 15 [ka] In a 100 mL reaction flask, intermediate 5 (5.0 g, 12.0 mmol), TsCl (11.4 g, 60.0 mmol) and DMAP (73 mg, 0.6 mmol) were added and dissolved in 50 mL of pyridine. The reaction was heated to 50°C and reacted at this temperature for 16 h. The reaction was monitored by TLC, and the developer was PE:EA=7:1. After the reaction was complete, the reaction solution was poured into 150 mL of water and stirred for 30 min. The reaction solution was filtered and the filter cake was washed with water to obtain 5.5 g of a white solid.

[0145] Step 5: Intermediate 16 [ka] TMSN3 (0.12 g, 1.1 eq, 2 eq), DBU (0.32 g, 0.21 mmol, 4 eq), and N2 were added dropwise to a suspension of intermediate 15 (0.3 g, 0.52 mmol, 1 eq) in DMF (6 mL) at 30-35 °C. The resulting reaction solution was heated to 80-90 °C and reacted for 16-20 h. When the intermediate 15 was almost completely consumed by TLC (PE:EA = 10:1), the reaction solution was cooled to 30-35 °C. The reaction solution was poured into water (20 mL) at 0-10 °C, stirred at 0-10 °C for 5-10 min, and then filtered. The filter cake was purified and eluted by silica gel column chromatography PE:EA to obtain 0.13 g of a solid, i.e., intermediate 16, with a yield of 56%.

[0146] Step 6: Intermediate 7 [ka] Under nitrogen protection, intermediate 16 (12.0 g, 27.2 mmol) and 100 mL of dichloromethane solvent were added to a flask. Furthermore, 4.8 g of Pd / C and 200 mL of ethanol were added. After hydrogen replacement (50 psi), the reaction system was heated to 50°C and reacted at this temperature for 16 h. The reaction was monitored by TLC, and the eluent was petroleum ether: ethyl acetate = 20:1. The reaction solution was filtered, and the filter cake was passed through a column (dichloromethane: methanol = 20:1) to obtain 11 g of a white solid, i.e., intermediate 7.

[0147] Step 7: Intermediate 17 [ka] Starting from intermediate 7, 40.4 g of intermediate 17 was obtained under the same conditions as described for intermediate 10.

[0148] Step 8: Intermediate 18 [ka] Using intermediate 17 as a raw material, intermediate 18 (35 g) was obtained under the same conditions as those described for intermediate 11.

[0149] Step 9: Example 13A [ka] 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 and reacted at room temperature for 16 h. The reaction was monitored by TLC thin layer chromatography (DCM / MeOH (5% ammonia methanol) = 20 / 1), and after completion of the reaction, the reaction solution was washed with water (250 mL), the aqueous phase was extracted with DCM (100 mL x 2), and the combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated, and purified by column (DCM / MeOH (5% ammonia methanol) = 45 / 1 to 15 / 1) to obtain about 17 g of the crude product of Example 13A. The crude product was dissolved in dichloromethane (about 120 mL), and then acetonitrile (200 mL) was added, and the solution was concentrated to about 100 mL under reduced pressure, and then stirred in an ice bath for 2 h, and a large amount of white solid precipitated. The reaction was filtered, the filter cake was dried, and then added to pure water (150 mL) at room temperature to make a slurry, stirred overnight, then filtered and dried to obtain Example 13A (12.2 g). NMR data: 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 = 3.7 Hz, 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.

[0150] Example 13B [ka]

[0151] Step 1: Intermediate 19 [ka] A mixture of intermediate 13 (240g, 0.42mol, 1eq) and 15-crown-5 (277.8g, 1.2mol, 3eq) in DMSO (12L) was stirred until completely dissolved, and NaN3 (81.9g, 1.26mol, 3eq) was added under N2 protection at 25-35°C. The resulting mixture was heated to 60-70°C and reacted for 2.5-3h, sampled under N2 protection, and monitored by TLC (PE:EA=9:1) that the raw material disappeared. After the reaction temperature dropped to 20-30°C, water was added, a solid precipitated, filtered, the filter cake was washed with 2L of water, and dried to obtain 177g of white solid, i.e., intermediate 19.

[0152] Step 2: Intermediate 23 [ka] Intermediate 19 (650 g) was dissolved in 10 L of DCM and 10 L of MeOH, and the solution was transferred to a pressure cooker. 104 g of 10% wet Pd / C was suspended in 200 mL of MeOH and transferred to a pressure cooker. At 28-35 °C, the pressure cooker was replaced with N2 four times, and then at 28-35 °C, the pressure cooker was replaced with H2 four times. Finally, the H2 pressure was maintained at 2.5-3 MPa, and the mixture was stirred at 28-35 °C for 48 h, sampled, and monitored by TLC (PE:EA = 20:1) before the raw material disappeared. After the reaction was completed, diatomaceous earth was spread on a suction filter funnel, and the reaction solution was suction filtered under reduced pressure to remove Pd / C, and the filter cake was subjected to column chromatography to obtain 460 g of a white solid, i.e., intermediate 9.

[0153] Step 3: Intermediate 20 [ka] Starting from intermediate 9, 40.4 g of intermediate 20 was obtained through the same experimental procedure as intermediate 10.

[0154] Step 4: Intermediate 21 [ka] Using intermediate 20 as a raw material, the same experimental procedure as for intermediate 11 was followed to give intermediate 21 (35 g) as a crude product.

[0155] Step 5: Example 13B [ka] 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 and reacted at room temperature for 16 h. The reaction was monitored by TLC thin layer chromatography (DCM / MeOH (5% ammonia methanol) = 20 / 1). After the reaction was completed, 250 mL of water was added, the aqueous phase was extracted with DCM (100 mL x 2), and the combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated, and then purified by column (DCM / MeOH (5% ammonia methanol) = 45 / 1 to 15 / 1) to obtain Example 13B (12.2 g). NMR data: 1H NMR (400 MHz, Chloroform-d) δ 7.39 (d, J = 8.2 Hz, 1H), 4.42 (td, J = 7.6, 6.0 Hz, 1H), 3.96 (dd, J = 11.0, 2.8 Hz, 1H), 3.87 - 3.70 (m, 1H), 3.65 (t, J = 5.2 Hz, 2H), 3.56 (q, J = 5.2 Hz, 2H), 3.31 (d, J = 11.0 Hz, 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 = 7.1 Hz, 4H), 1.04 (d, J = 3.3 Hz, 1H), 1.00 (d, J = 6.5 Hz, 3H), 0.94 (s, 3H), 0.75 (s, 3H). Mass spectrum: [M+1]641.5.

[0156] Example 14 [ka]

[0157] Procedure 1: Example 14 [ka] 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, dissolved in 3 mL of dry DCM, stirred at room temperature, reacted for 1 h, and monitored for reaction completion (PMA coloring) by TLC. After completion of the reaction, the mixture was washed twice with saturated aqueous NH4Cl, dried, and purified by thin layer chromatography (dichloromethane:methanol=10:1) to obtain 38 mg of white solid example 14. NMR data: 1HNMR(CDCl3, 400MHz, 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 spectrum:[M+1]545.5

[0158] Example 14A [ka] Starting from Intermediate 18, the same synthesis method as in Example 14 can be used to obtain Example 14A as a white solid. NMR data: 1H NMR (400 MHz, Chloroform-d) δ 7.21 (s, 1H), 7.07 (d, J = 7.7 Hz, 1H), 4.41 (q, J = 7.5 Hz, 1H), 4.18 (s, 1H), 3.95 (dd, J = 11.0, 2.8 Hz, 1H), 3.74 (q, J = 5.1 Hz, 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 spectrum: [M+1]545.5.

[0159] Example 14B [ka] Starting from intermediate 21, the same synthesis method as in Example 14 can be used to obtain Example 14B as a white solid. NMR data: 1H NMR (400 MHz, Chloroform-d) δ 7.42 (s, 1H), 6.67 (s, 1H), 4.41 (td, J = 7.8, 6.2 Hz, 1H), 3.96 (dd, J = 11.0, 2.8 Hz, 1H), 3.84-3.66 (m, 3H), 3.44 (td, J = 5.6, 4.4 Hz, 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), 1.50-1.31 (m, 7H), 1.31-1.13 (m, 6H), 1.08 (m, 3H), 1.00 (d, J = 6.6 Hz, 3H), 0.95 (s, 3H), 0.75 (s, 3H). Mass spectrum: [M+1]545.5.

[0160] Example 15 [ka] Example 15 was obtained in the same manner as in Example 14 using isothymosarsasapogenin as a raw material. NMR data: 1H NMR (400 MHz, Chloroform-d) δ7.48 (s, 1H), 7.40 (d, J = 7.0 Hz, 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 = 14.7 Hz, 2H), 3.07 (q, J = 7.4 Hz, 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, 11.9 Hz, 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, 1.5 Hz, 3H), 0.75 (d, J = 1.8 Hz, 3H). Mass spectrum: [M+1]545.5.

[0161] Example 15A [ka] Example 15A was obtained using isothymosarsasapogenin as the raw material in the same manner as in Example 14A. NMR data: 1H NMR (400 MHz, Chloroform-d) δ 7.40 (s, 1H), 7.30 (d, J = 7.6 Hz, 1H), 4.40 (td, J = 7.7, 5.9 Hz, 1H), 4.17 (s, 1H), 3.73 (d, J = 4.8 Hz, 2H), 3.41 (m, 4H), 3.20 (s, 2H), 2.92 (s, 1H), 1.43 (m, 33H), 0.78 (m, 6H). Mass spectrum: [M+1]545.5.

[0162] Example 15B [ka] Example 15B was obtained using isothymosarsasapogenin as the raw material in the same manner as in Example 14A. NMR data: 1H NMR (400 MHz, Chloroform-d) δ 7.51 (s, 1H), 6.83 (d, J = 7.9 Hz, 1H), 4.41 (q, J = 7.4 Hz, 1H), 3.74 (m, 3H), 3.42 (m, 4H), 3.17 (s, 2H), 1.38 (m, 34H), 0.78 (m, 6H). Mass spectrum: [M+1]545.5.

[0163] Example 16A [ka] Example 16A was obtained using isothymosarsasapogenin as the raw material in the same manner as in Example 13A. NMR data: 1H NMR (400 MHz, Chloroform-d) δ 8.02 (d, J = 7.7 Hz, 1H), 4.46-4.33 (m, 1H), 4.18 (d, J = 7.3 Hz, 1H), 3.66 (dq, J = 5.7, 3.1, 2.2 Hz, 2H), 3.57 (q, J = 5.2, 4.6 Hz, 2H), 3.47 (ddd, J = 10.9, 4.4, 2.0 Hz, 1H), 3.37 (t, J = 10.9 Hz, 1H), 3.30 (d, J = 3.4 Hz, 2H), 2.52 (d, J = 2.9 Hz, 4H), 2.48 (t, J = 6.1 Hz, 4H), 2.33 (d, J = 6.0 Hz, 7H), 2.07-1.28 (m, 29H), 1.23-1.02 (m, 10H), 1.00 (s, 3H), 0.96 (d, J = 6.9 Hz, 4H), 0.92-0.81 (m, 5H), 0.79 (d, J = 6.3 Hz, 3H), 0.76 (s, 3H). Mass spectrum: [M+1]641.5.

[0164] Example 16B [ka] Using isothymosarsasapogenin as the raw material, Example 16B was obtained through the same preparation procedure and under the same conditions as Example 13B. NMR data: 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 spectrum: [M+1] 641.5.

[0165] Example 17A [ka] Starting from isosarsasapogenin, intermediate 22 was obtained through the same preparation procedure and under the same conditions as intermediate 7. Starting from intermediate 22, example 17A was obtained through the same preparation method and conditions as procedure 5 of example 1A. Mass spectrum: [M+1] 570.

[0166] Example 17B [ka] Using isosarsasapogenin as a raw material, intermediate 23 was obtained through the same preparation procedure and under the same conditions as intermediate 9. Using intermediate 23 as a raw material, example 17B was obtained through the same preparation method and conditions as example 1B. Mass spectrum: [M+1] 570.

[0167] Example 18A [ka] Using isosarsasapogenin as the raw material, Example 18A was obtained using the same preparation procedures and conditions as Example 4A. NMR data: 1H NMR (400 MHz, 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, 5.2 Hz, 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 spectrum: [M+1]515.

[0168] Example 18B [ka] Using isosarsasapogenin as the raw material, Example 18B was obtained using the same preparation procedures and conditions as Example 4B. NMR data: 1H NMR (400 MHz, 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 = 4.3 Hz, 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 = 6.3 Hz, 3H), 0.75 (s, 3H). Mass spectrum: [M+1]515.

[0169] Example 19A [ka] Using hecogenin as a raw material, intermediate 24 was obtained through the same preparation procedure and under the same conditions as intermediate 7. Using intermediate 24 as a raw material, example 19A was obtained through the same preparation method and conditions as procedure 5 of example 1A. NMR data: 1H NMR (400 MHz, Chloroform-d) δ 4.33 (t, J = 7.4 Hz, 1H), 3.79-3.61 (m, 3H), 3.50 (s, 2H), 3.35 (t, J = 10.9 Hz, 2H), 3.10 (q, J = 7.4 Hz, 2H), 2.99 (s, 7H), 2.70 (s, 1H), 2.62 -2.45 (m, 5H), 2.38 (t, J = 13.7 Hz, 2H), 2.22 (dd, J = 14.3, 5.0 Hz, 2H), 2.11 (q, J = 6.5, 6.0 Hz, 2H), 1.90 (d, J = 9.0 Hz, 3H), 1.75 (dt, J = 12.9, 6.5 Hz, 5H), 1.69-1.50 (m, 11H), 1.44 (dd, J = 11.3, 7.9 Hz, 6H), 1.40-1.09 (m, 10H), 1.09-1.01 (m, 5H), 0.97 (dd, J = 12.9, 7.9 Hz, 2H), 0.89 (s, 4H), 0.79 (d, J = 6.3 Hz, 3H). Mass spectrum: [M+1]529.4.

[0170] Example 20A [ka] Example 20A was obtained using hecogenin as the raw material and the same preparation procedures and conditions as those for Example 4A. NMR data: 1H NMR (400 MHz, Chloroform-d) δ 6.99 (t, J = 6.4 Hz, 1H), 4.33 (dd, J = 8.5, 5.8 Hz, 1H), 3.81-3.57 (m,1H), 3.57-3.43 (m, 1H), 3.35 (t, J = 11.0 Hz, 1H), 3.15 (t, J = 6.5 Hz, 2H), 2.71 (t, J = 6.5 Hz, 2H), 2.66 (s, 6H), 2.51 (dd, J = 8.8, 6.7 Hz, 1H), 2.38 (t, J = 13.7 Hz, 1H), 2.21 (dd, J = 14.3, 5.0 Hz, 1H), 2.11 (t, J = 7.1 Hz, 1H), 1.89 (dd, J = 11.1, 7.3 Hz, 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 = 6.3 Hz, 3H). Mass spectrum: [M+1]529.4.

[0171] Example 21 [ka] Example 21 was obtained using 4-hydroxyphenethylamine as the raw material and the same preparation procedure and conditions as Example 13. NMR data: 1HNMR (CDCl3, 400MHz, 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 spectrum: [M+1]621.4.

[0172] Example 22 [ka] Example 22 was obtained using 4-dihydroxyphenethylamine as the raw material and the same preparation procedure and conditions as Example 13. NMR data: 1HNMR (CDCl3, 400MHz, ppm): 0.75 (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 spectrum: [M+1]637.4.

[0173] Example 23 [ka] The preparation procedure is as follows:

[0174] Step 1: Intermediate 25 [ka] Under argon gas protection, 0.2g of morpholine, 0.4g of 1-Boc-3-azetidinone, 0.7mL of acetic acid, and 0.3g of sodium cyanoborohydride were dissolved in 20mL of dichloromethane and reacted at room temperature for 2h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was spin-dried. The product was purified by silica gel column chromatography DCM:MeOH=20:1, and detected by thin layer chromatography DCM:MeOH=10:1. The product was colored with phosphomolybdic acid. The yield was 70%.

[0175] Step 2: Intermediate 26 [ka] Under argon gas protection, 0.2g of intermediate 25 was dissolved in 10mL of dichloromethane, 5mL of dioxane hydrochloride solution was added, and the reaction was carried out at room temperature for 2h, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and the color was developed with phosphomolybdic acid. The yield was 90%.

[0176] Step 3: Synthesis of Example 23 [ka] Under argon gas protection, 0.1g of intermediate 26, 0.43g of intermediate 11, 0.40g of HATU and 0.22g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, the solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with phosphomolybdic acid. The yield was 60%. NMR data: 1H NMR (400 MHz, 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)., 3.62-3.70 (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 spectrum: [M+1] 626.5.

[0177] Example 24 [ka] The preparation procedure is as follows:

[0178] [ka] Under argon gas protection, 0.2g of intermediate 11, 0.08g of 1-methyl-4-(4-piperidinyl)piperazine, 0.227g of HATU and 0.101g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, and the solvent was spin-dried. The product was purified by silica gel column chromatography DCM:MeOH=20:1, and detected by thin layer chromatography DCM:MeOH=10:1. The product was colored with vanillin. The yield was 45%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]667.5.

[0179] Example 24A, Example 24B [ka] Starting from intermediate 18, example 24A can be obtained by the same synthesis method as in example 24. Starting from intermediate 21, example 24B can be obtained by the same method. NMR data for Example 24A: 1H NMR (400 MHz, 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 spectrum: [M+1] 667.5. NMR data of Example 24B: 1H NMR (400 MHz, 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 spectrum: [M+1]667.5.

[0180] Example 25 [ka] The preparation procedure is as follows:

[0181] [ka] The preparation method is the same as in Example 24. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]681.6.

[0182] Example 25A, Example 25B [ka] Starting from intermediate 18 and 1-ethyl-4-(piperidin-4-ylmethyl)piperazine, Example 25A can be obtained by the same synthesis method as in Example 24. Starting from intermediate 21 and 1-ethyl-4-(piperidin-4-ylmethyl)piperazine, Example 25B can be obtained by the same synthesis method. NMR data of Example 25A: 1H NMR (400 MHz, 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 spectrum: [M+1]681.6. NMR data for Example 25B: 1H NMR (400 MHz, 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 spectrum: [M+1] 681.6.

[0183] Example 26 [ka] The preparation procedure is as follows:

[0184] [ka] The preparation method is the same as in Example 24. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]667.6.

[0185] Example 26A, Example 26B [ka] Example 26A can be obtained by the same synthesis method as Example 24 using intermediate 18 and 1-(1-methyl-4-piperidinyl)piperazine as starting materials. Example 26B can be obtained by the same synthesis method as Example 24 using intermediate 21 and 1-(1-methyl-4-piperidinyl)piperazine as starting materials. NMR data of Example 26A: 1H NMR (400 MHz, 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 spectrum: [M+1]667.6. NMR data for Example 26B: 1H NMR (400 MHz, 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 spectrum: [M+1] 667.6.

[0186] Example 27 [ka] The preparation procedure is as follows:

[0187] Step 1: Intermediate 27 [ka] 0.5g of 4-(2-aminoethyl)piperazine-1-carboxylic acid tert-butyl ester and 0.714g of di-tert-butyl dicarbonate were dissolved in 20 mL of dry DCM and reacted at room temperature for 12h, and the reaction was detected. After the reaction was complete, the solvent was spin-dried, extracted with water / dichloromethane, purified by silica gel column chromatography (DCM:MeOH=30:1), detected by thin layer chromatography (DCM:MeOH=10:1), and colored with vanillin. The yield was 65%.

[0188] Step 2: Intermediate 28 [ka] 0.2g of intermediate 27 and 0.07g of LiAlH4 were added to 15 mL of THF, heated to reflux, and reacted for 3h. The reaction was detected. After the reaction was complete, it was cooled to room temperature, the solvent was spin-dried, and the product was obtained, which was directly put into the next reaction and colored with vanillin. The yield was 56%.

[0189] Step 3: Synthesis of Example 27 [ka] Under argon gas protection, 0.037g of intermediate 28, 0.12g of intermediate 11, 0.136g of HATU and 0.061g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, the solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with vanillin. The yield was 55%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]641.6.

[0190] Example 27A, Example 27B [ka] Its synthesis is the same as that of Example 27, specifically, as shown below, intermediate 18 and N-methyl-2-(1-methylpiperidin-4-yl)ethan-1-amine are used as raw materials, and Example 27A can be obtained by the same synthesis method as that of Example 27. Example 27A was obtained by the same method as that of Example 27 using intermediate 21 and N-methyl-2-(1-methylpiperidin-4-yl)ethan-1-amine as raw materials. NMR data of Example 27A: 1H NMR (400 MHz, 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 spectrum: [M+1]641.6. NMR data of Example 27B: 1H NMR (400 MHz, 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 spectrum: [M+1]641.6.

[0191] Example 28 [ka] The preparation procedure is as follows:

[0192] [ka] Under argon gas protection, 0.1g of piperazine-1-carboxylic acid methyl ester, 0.42g of intermediate 11, 0.40g of HATU and 0.21g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, and the solvent was spin-dried. The product was purified by silica gel column chromatography DCM:MeOH=20:1, and detected by thin layer chromatography DCM:MeOH=10:1. The product was colored with phosphomolybdic acid. The yield was 65%. NMR data: 1H NMR (CDCl3-d6, 400 MHz): 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 spectrum: [M+1] 628.5.

[0193] Example 29 [ka] The preparation procedure is as follows:

[0194] Step 1: Intermediate 29 [ka] Under argon gas protection, 0.1g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.05g of bromoethane, and 0.08g of TEA were dissolved in 30 mL of dichloromethane and reacted at room temperature for 12 hours, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and colored with phosphomolybdic acid. The yield was 80%.

[0195] Step 2: Intermediate 30 [ka] Under argon gas protection, 0.1g of intermediate 29 was dissolved in 10mL of dichloromethane, 5mL of dioxane hydrochloride solution was added, and the reaction was carried out at room temperature for 2h, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and the color was developed with phosphomolybdic acid. The yield was 90%.

[0196] Step 3: Synthesis of Example 29 [ka] Under argon gas protection, 0.05g of intermediate 30, 0.15g of intermediate 11, 0.15g of HATU and 0.08g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, the solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with phosphomolybdic acid. The yield was 60%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]681.5.

[0197] Example 29A, Example 29B [ka] The synthesis process is the same as that of Example 29. Example 29A can be obtained from intermediate 18 and 1-(1-ethylpiperidin-4-yl)piperazine by the same synthesis method as that of Example 29. Example 29B can be obtained from intermediate 21 and 1-(1-ethylpiperidin-4-yl)piperazine by the same synthesis method as that of Example 29. NMR data of Example 29A: 1H NMR (400 MHz, 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 spectrum: [M+1] 681.6. NMR data of Example 29B: 1H NMR (400 MHz, 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 spectrum: [M+1] 681.5.

[0198] Example 30 [ka] The preparation procedure is as follows:

[0199] Step 1: Intermediate 31 [ka] Under argon gas protection, 0.4g of intermediate 11, 0.258g of 4-(piperidin-4-yl)piperazine-1-carboxylic acid tert-butyl ester, 0.455g of HATU, and 0.202g of TEA were dissolved in 15 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was spin-dried. The product was purified by silica gel column chromatography DCM:MeOH=20:1, and detected by thin layer chromatography DCM:MeOH=10:1. The product was colored with vanillin, and the yield was 75%.

[0200] Step 2: Intermediate 32 [ka] 0.2 g of intermediate 31 was added to 10 mL of MeOH, and 10 mL of 4M HCl / dioxane solution was added and reacted at room temperature for 3 h. The solvent was spin-dried and the mixture was directly put into the next reaction and colored with vanillin. The yield was 80%.

[0201] Step 3: Synthesis of Example 30 [ka] Under argon gas protection, 0.08g of intermediate 32, 0.03g of bromoacetonitrile, 0.034g of potassium carbonate, and 0.02g of potassium iodide were dissolved in 10 mL of acetonitrile, heated to 50°C, and reacted for 12h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was spin-dried. The product was purified by silica gel column chromatography DCM:MeOH=20:1, and detected by thin layer chromatography DCM:MeOH=10:1. The product was colored with vanillin, and the yield was 65%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]693.6.

[0202] Example 31 [ka] The preparation procedure is as follows:

[0203] Step 1: Intermediate 33 [ka] 0.1g of 4-(piperidin-4-yl)piperazine-1-carboxylic acid tert-butyl ester and 0.089g of bromoacetonitrile were dissolved in 10 mL of dry MeCN, and 0.103g of potassium carbonate and 0.062g of potassium iodide were added. The mixture was heated to 50°C and reacted for 12h, and the reaction was detected. After the reaction was complete, the mixture was extracted with water / dichloromethane, purified by silica gel column chromatography (DCM:MeOH=30:1), detected by thin layer chromatography (DCM:MeOH=10:1), and colored with vanillin. The yield was 55%.

[0204] Step 2: Intermediate 34 [ka] 0.2g of intermediate 33 was added to 10mL of MeOH, and 10mL of 4M HCl / dioxane solution was added and reacted at room temperature for 3h. The solvent was spin-dried and the mixture was directly put into the next reaction and colored with vanillin. The yield was 80%.

[0205] Step 3: Synthesis of Example 31 [ka] Under argon gas protection, 0.046g of intermediate 34, 0.1g of intermediate 11, 0.114g of HATU and 0.061g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, the solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with vanillin. The yield was 40%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1] 692.6.

[0206] Example 32 [ka] The preparation procedure is as follows:

[0207] Step 1: Intermediate 35 [ka] Under argon gas protection, 0.1g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.06g of bromoisobutane, and 0.08g of TEA were dissolved in 30 mL of N,N-dimethylformamide and heated to 50°C to detect the reaction. The solvent was spin-dried and directly put into the next reaction, where it was colored with phosphomolybdic acid. The yield was 70%.

[0208] Step 2: Intermediate 36 [ka] Under argon gas protection, 0.1g of intermediate 35 was dissolved in 10mL of dichloromethane, 5mL of dioxane hydrochloride solution was added, and the reaction was carried out at room temperature for 2h, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and the color was developed with phosphomolybdic acid. The yield was 90%.

[0209] Step 3: Synthesis of Example 36 [ka] Under argon gas protection, 0.05g of intermediate 36, 0.11g of intermediate 11, 0.13g of HATU and 0.07g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, the solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with phosphomolybdic acid. The yield was 60%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]709.7.

[0210] Example 32A, Example 32B [ka] The synthesis method is the same as for 32.

[0211] Starting from intermediate 18 and 1-(1-isobutylpiperidin-4-yl)piperazine, Example 32A can be obtained by the same synthesis method as in Example 32. Starting from intermediate 21 and 1-(1-isobutylpiperidin-4-yl)piperazine, Example 32B can be obtained by the same synthesis method. NMR data of Example 32A: 1H NMR (400 MHz, 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 spectrum: [M+1]709.7. NMR data of Example 32B: 1H NMR (400 MHz, 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 spectrum: [M+1]709.7.

[0212] Example 33 [ka] The preparation procedure is as follows:

[0213] Step 1: Intermediate 37 [ka] Under argon gas protection, 0.1g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.22g of intermediate 11, 0.21g of HATU and 0.11g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was spin-dried. The product was purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with phosphomolybdic acid. The yield was 60%.

[0214] Step 2: Intermediate 38 [ka] Under argon gas protection, 0.1g of intermediate 37 was dissolved in 10mL of dichloromethane, 5mL of dioxane hydrochloride solution was added, and the reaction was carried out at room temperature for 2h, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and the color was developed with phosphomolybdic acid. The yield was 90%.

[0215] Step 3: Synthesis of Example 33 [ka] Under argon gas 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, heated to 50 °C, and reacted for 12 h. The reaction was detected. The solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH = 20:1, and detected by thin layer chromatography DCM:MeOH = 10:1. The color was developed with phosphomolybdic acid, and the yield was 60%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]709.6.

[0216] Example 33A, Example 33B [ka] The synthesis process is as follows. Using intermediate 18 and 1-bromo-2-methylpropane as raw materials, Example 33A can be obtained by the same synthesis method as Example 33. Using intermediate 21 and 1-bromo-2-methylpropane as raw materials, Example 33B can be obtained by the same method. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]709.6. NMR data of Example 33B: 1H NMR (400 MHz, 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 spectrum: [M+1]709.5.

[0217] Example 34 [ka] The preparation procedure is as follows:

[0218] Step 1: Intermediate 39 [ka] Under argon gas protection, 0.1g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.06g of 2-bromopropane, and 0.08g of TEA were dissolved in 30 mL of N,N-dimethylformamide and heated to 50°C to detect the reaction. The solvent was spin-dried and directly put into the next reaction, and colored with phosphomolybdic acid. The yield was 70%.

[0219] Step 2: Intermediate 40 [ka] Under argon gas protection, 0.1g of intermediate 39 was dissolved in 10mL of dichloromethane, 5mL of dioxane hydrochloride solution was added, and the reaction was carried out at room temperature for 2h, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and the color was developed with phosphomolybdic acid. The yield was 90%.

[0220] Step 3: Synthesis of Example 34 [ka] Under argon gas protection, 0.05g of intermediate 40, 0.14g of intermediate 11, 0.13g of HATU and 0.07g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, the solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with phosphomolybdic acid. The yield was 60%. NMR data: 1H NMR (400 MHz, 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, Mass spectrum: [M+1]695.7.

[0221] Example 34A, Example 34B [ka] The synthesis process is as follows. Example 34A can be obtained from intermediate 18 and 1-(1-isopropylpiperidin-4-yl)piperazine in the same synthesis method as in Example 34. Example 34B can be obtained from intermediate 21 and 1-(1-isopropylpiperidin-4-yl)piperazine in the same synthesis method. NMR data for Example 34A: 1H NMR (400 MHz, 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 spectrum: [M+1] 695.7. NMR data for Example 34B: 1H NMR (400 MHz, 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 spectrum: [M+1] 695.6.

[0222] Example 35 [ka] The preparation procedure is as follows:

[0223] [ka] Example 35 can be obtained by the same synthesis method as Example 28 using intermediate 11 and 1-(pyridin-4-yl)piperazine as starting materials. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]647.5.

[0224] Example 35A, Example 35B [ka] The synthesis process is as follows. Example 35A can be obtained by the same synthesis method as Example 28 using intermediate 18 and 1-(pyridin-4-yl)piperazine as raw materials. Example 35B can be obtained by the same synthesis method as Example 28 using intermediate 21 and 1-(pyridin-4-yl)piperazine as raw materials. NMR data of Example 35A: 1H NMR (400 MHz, 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 spectrum: [M+1]647.6. NMR data of Example 35B: 1H NMR (400 MHz, 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 spectrum: [M+1]647.5.

[0225] Example 36 [ka] The preparation procedure is as follows:

[0226] [ka] Example 36 can be obtained by the same synthesis method as Example 31 using intermediate 11 and 1-(1-(2-fluoroethyl)piperidin-4-yl)piperazine as starting materials. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]699.6.

[0227] Example 37 [ka] The preparation procedure is as follows:

[0228] Step 1: Intermediate 41 [ka] Under argon gas protection, 0.2g of N,N-dimethylglycine, 0.36g of 1-tert-butoxycarbonylpiperazine, 1.1g of HATU and 0.6g of TEA were dissolved in 60 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, and the solvent was spin-dried. The product was purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with phosphomolybdic acid. The yield was 80%.

[0229] Step 2: Intermediate 42 [ka] Under argon gas protection, 0.2g of intermediate 41 was dissolved in 10mL of dichloromethane, 5mL of dioxane hydrochloride solution was added, and the reaction was carried out at room temperature for 2h, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and the color was developed with phosphomolybdic acid. The yield was 90%.

[0230] Step 3: Synthesis of Example 37 [ka] Under argon gas protection, 0.05g of intermediate 42, 0.18g of intermediate 11, 0.17g of HATU and 0.1g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, the solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with phosphomolybdic acid. The yield was 60%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]655.6.

[0231] Example 38 [ka] The preparation procedure is as follows:

[0232] Step 1: Intermediate 43 [ka] Under argon gas protection, 0.1g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.1g of 2-bromo-N,N-dimethylacetamide, and 0.08g of TEA were dissolved in 30 mL of dichloromethane and reacted at room temperature for 12 hours, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and colored with phosphomolybdic acid. The yield was 80%.

[0233] Step 2: Intermediate 44 [ka] Under argon gas protection, 0.1g of intermediate 43 was dissolved in 10mL of dichloromethane, 5mL of dioxane hydrochloride solution was added, and the reaction was carried out at room temperature for 2h, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and the color was developed with phosphomolybdic acid. The yield was 90%.

[0234] Step 3: Synthesis of Example 38 [ka] Under argon gas protection, 0.05g of intermediate 44, 0.18g of intermediate 11, 0.17g of HATU and 0.09g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, the solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with phosphomolybdic acid. The yield was 60%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]655.6.

[0235] Example 39 [ka] The preparation procedure is as follows:

[0236] Step 1: Intermediate 45 [ka] Under argon gas protection, 0.1g of N,N-dimethylaminoethyl bromide hydrobromide, 0.08g of 1,4-diazepine-1-carboxylic acid tert-butyl ester, and 0.13g of TEA were dissolved in 30 mL of dichloromethane and reacted at room temperature for 12 hours, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and colored with phosphomolybdic acid. The yield was 80%. Step 2: Intermediate 46 [ka] Under argon gas protection, 0.1g of intermediate 45 was dissolved in 10mL of dichloromethane, 5mL of dioxane hydrochloride solution was added, and the reaction was carried out at room temperature for 2h, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and the color was developed with phosphomolybdic acid. The yield was 90%.

[0237] Step 3: Synthesis of Example 39 [ka] Under argon gas protection, 0.05g of intermediate 46, 0.18g of intermediate 11, 0.17g of HATU and 0.09g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, the solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with phosphomolybdic acid. The yield was 60%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]655.6.

[0238] Example 40 [ka] The preparation procedure is as follows:

[0239] Step 1: Intermediate 47 [ka] Under argon gas protection, 0.2g of imidazole, 0.5g of 1-bromo-2-chloroethane, and 0.6g of TEA were dissolved in 30 mL of dichloromethane and reacted at room temperature for 12 hours, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and colored with phosphomolybdic acid. The yield was 80%.

[0240] Step 2: Intermediate 48 [ka] Under argon gas protection, 0.1g of 1-Boc-4-(piperidin-4-yl)-piperazine, 0.08g of intermediate 47 and 0.08g of TEA were dissolved in 30 mL of dichloromethane and reacted at room temperature for 12 hours, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and colored with phosphomolybdic acid. The yield was 70%.

[0241] Step 3: Intermediate 49 [ka] Under argon gas protection, 0.1g of intermediate 48 was dissolved in 10mL of dichloromethane, 5mL of dioxane hydrochloride solution was added, and the reaction was carried out at room temperature for 2h, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and the color was developed with phosphomolybdic acid. The yield was 90%.

[0242] Step 4: Synthesis of Example 40 [ka] Under argon gas protection, 0.05g of intermediate 49, 0.17g of intermediate 11, 0.16g of HATU and 0.08g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, the solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with phosphomolybdic acid. The yield was 60%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1] 664.6.

[0243] Example 41 [ka] The preparation procedure is as follows:

[0244] Step 1: Intermediate 50 [ka] Under argon gas protection, 0.2g of 2-diethylamino-1-bromoethane hydrobromide and 0.15g of 1-tert-butoxycarbonylpiperazine were dissolved in 40mL of acetonitrile and reacted at 50℃ for 12h, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and colored with phosphomolybdic acid. The yield was 80%.

[0245] Step 2: Intermediate 51 [ka] Under argon gas protection, 0.1g of intermediate 50 was dissolved in 10mL of dichloromethane, 5mL of a solution of hydrochloric acid in ethyl acetate was added, and the reaction was carried out at room temperature for 12h, and the reaction was detected. The solvent was spin-dried and directly put into the next reaction, and the color was developed with phosphomolybdic acid. The yield was 90%.

[0246] Step 3: Synthesis of Example 41 [ka] Under argon gas protection, 0.05g of intermediate 51, 0.14g of intermediate 11, 0.15g of HATU and 0.08g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried with anhydrous sodium sulfate, the solvent was spin-dried, purified by silica gel column chromatography DCM:MeOH=20:1, detected by thin layer chromatography DCM:MeOH=10:1, and colored with phosphomolybdic acid. The yield was 60%. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1]669.7.

[0247] Example 41A, Example 41B [ka] The synthetic process of Example 41A was the same as that of Example 41, by replacing intermediate 11 with intermediate 18. NMR data: 1H NMR (CDCl3-d6, 400 MHz): 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 spectrum: [M+1] 669.7. The synthetic process of Example 41B was the same as that of Example 41, by replacing intermediate 11 with intermediate 21. NMR data: 1H NMR (CDCl3-d6, 400 MHz): 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), 3.28 (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 spectrum: [M+1] 669.6.

[0248] Example 42 [ka] The preparation procedure is as follows:

[0249] Step 1: Intermediate 52 [ka] Under argon gas protection, 1g of intermediate 3, 0.933g of 3-(1-tert-butoxycarbonylpiperazin-4-yl)propionic acid, 1.373g of HATU, and 0.788g of DIPEA were dissolved in 20 mL of dichloromethane and reacted at room temperature for 6 h. The reaction was detected. The product was extracted with water / dichloromethane, washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was spin-dried. The product was purified by silica gel column chromatography DCM:MeOH=20:1, and detected by thin layer chromatography DCM:MeOH=10:1. The product was colored with vanillin, and the yield was 65%.

[0250] Step 2: Intermediate 53 [ka] 1g of intermediate 52 was dissolved in 10 mL of MeOH, and 4M HCl / dioxane was added and reacted for 3h. The reaction was detected. After the reaction was complete, the solvent was spun dry to obtain the product, which was directly put into the next reaction and colored with vanillin. The yield was 80%.

[0251] Step 3: Synthesis of Example 42 [ka] Under argon gas protection, 0.2g of intermediate 53, 0.077g of 3-pyrrolidin-1-yl-propionic acid, 0.205g of HATU and 0.091g of TEA were dissolved in 10 mL of dichloromethane and reacted at room temperature for 6h. The reaction was detected. It was extracted with water / dichloromethane, washed with saturated saline, dried over anhydrous sodium sulfate, the solvent was spin-dried, and purified by silica gel column chromatography DCM:MeOH=20:1, and colored with vanillin. The yield was 60%. NMR data: 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 spectrum: [M+1]682.6.

[0252] Example 42A [ka] The preparation process was the same as that of Example 42, by replacing Intermediate 3 with Intermediate 7 to obtain Example 42A. NMR data: 1H NMR (400 MHz, 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 spectrum: [M+1] 682.7.

[0253] Example 43 Compound Activity Test Experiment 1. Measurement of the protective effect of small molecule compounds against injury of primary cultured rat neurons induced by oxygen glucose deprivation (OGD) model Experimental and analytical methods: Rats on the 15th day of pregnancy were purchased, and fetal rat brains (E15-16) were dissected. Primary culture of cerebral cortical neurons was performed in neuronal culture medium for 14 days (DIV 14) in a 48-well plate for OGD experiments. During the OGD experiment, the neuronal culture medium was changed to a sugar-free anaerobic medium (95% N2 / 5% CO2 balance), and the neurons were subjected to OGD treatment in an OGD chamber. Then, the medium was changed to a normal neuronal medium and placed in a 95% Air / 5% CO2 incubator for 24 h of culture, after which neuronal activity analysis was performed. The wells were stained with Hoechst 33342 and propidium iodide, and photographed under a high-content fluorescence microscope. For each replicate well, 20 non-overlapping fields were photographed randomly (Thermo Scientific TM The total number of neurons and the number of dead neurons were counted using the HCS Studio Cell Analysis Software. The standard deviation and standard error were calculated from the obtained data, and the P value was calculated by T-test analysis to determine whether there was a significant difference. Compound treatment: 13 kinds of small molecule compounds from Examples 1 to 20 were diluted with DMSO to 0.5 mM mother solution, and 1 μL of mother solution was added to 0.5 ml of medium in each well of a 48-well plate so that the final concentration was 1 μM. 1 μL of the positive control compound DPQ (10 mM mother solution) and 1 μL of the negative control DMSO were added to the medium 24 hours before the OGD experiment, and were also added at the same concentrations to the sugar-free anaerobic medium and the normal neuronal medium thereafter. Results: As shown in Figure 1, the results of this OGD experiment showed that the percentage of neuronal death without OGD was 10.32%, the percentage of neuronal death in the negative control with DMSO was 52.31%, and the injury rate was about 42%. The percentage of neuronal death in the positive control with DPQ was 22.22%, showing a highly significant protective effect against neuronal injury.

[0254] Experiment 2. Measurement of the protective effect of the small molecule compounds of Examples 1 to 14B against oxidative damage induced by hydrogen peroxide (H2O2) in human SHSY5Y neuronal tumor cells [Table 1]

[0255] [Table 2]

[0256] Results: As shown in Figure 2, Examples 2, 7, 13, and 14A showed significant protective effects against hydrogen peroxide-induced neuronal cell damage, among which undifferentiated SH-SY5Y cells were pre-protected for 24 h and treated with hydrogen peroxide at a final concentration of 1 μM, and then measured 24 hours later.

[0257] Experiment 3. Measurement of the protective effect of small molecule compounds against L-cysteine-induced neuronal excitotoxicity Experimental and analytical methods: Before the experiment, 24-well plates were pretreated with poly-D-lysine and cultured overnight in a cell incubator at 37°C and 5% CO2. SD rats on the 18th day of pregnancy were purchased, and the rat fetal brains (E18-19) were dissected. Using 24-well plates, primary culture of hippocampal neurons was performed in neuronal medium, and hippocampal neurons were cultured in serum-free medium. After 19 days of culture (DIV19) in a cell incubator at 37°C and 5% CO2, the test compound (final concentration 1 μM) was added, and the positive control was AP5 [(2R)-amino-5-phosphonopentanoic acid (ester)] (final concentration 100 μM). 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 to stain the cells. The cells were cultured in an incubator for 15 min and then photographed under a high-content fluorescence microscope. Six non-overlapping fields were randomly photographed for each replicate well, and the total number of neurons and the number of dead neurons were counted using Image J software. The data obtained were used to determine whether there was a difference between the drug-treated group and the control group. Compound treatment: 32 small molecule compounds from Examples 1 to 19A were diluted with DMSO to 1 mM mother solution, and 1 μL of mother solution was added to 1 ml of medium in each well of a 24-well plate to give a final concentration of 1 μM. The positive control compound was 100 μl AP5 (100 mM mother solution), and the negative control was 1 μl DMSO. Results: As shown in Figure 3, the percentage of neuronal death cells without L-cystine (400μM) and NaHCO3 (10mM) was 20.12%, while the percentage of neuronal death cells in the negative control with DMSO was 87.62%, with the damage rate being about 67.5%. The percentage of neuronal death cells in the positive control with AP5 was 27.58%, showing that AP5 had a highly significant protective effect against neuronal excitotoxicity. The experimental results of the four small molecule compounds showed the protective effects of compounds Example 1, Example 1A, Example 2, Example 4B, Example 6, Example 10, Example 13, Example 13B, etc. against neuronal excitotoxicity induced by L-cystine (400μM).

[0258] Experiment 4. Evaluation of the anti-inflammatory effects of "Example 5" and "Example 6" in zebrafish Experimental animals Transgenic neutrophil fluorescent zebrafish were adopted and natural pair mating breeding was performed. The age was 3 days post-fertilization (3dpf), and there were 30 in each experimental group, totaling 810. They were used to determine the minimum toxic concentration (MTC) of "Example 5" and "Example 6" in the LPS-induced inflammation experiment, and to evaluate the anti-inflammatory effect of "Example 5" and "Example 6" against LPS-induced inflammation. Zebrafish were provided by our aquaculture center and kept in aquaculture water at 28°C (water quality: 200 mg of instant sea salt was added per 1 L of reverse osmosis water, conductivity was 480-510 μS / cm, pH was 6.9-7.2, and hardness was 53.7-71.6 mg / L CaCO3). Laboratory animal use permit number: SYXK(Zhejiang)2012-0171. The breeding management complied with the requirements of the international AAALAC certification.

[0259] Experimental Agents "Example 5" is a white powder stored dry at 4 ° C. and was supplied by Shenzhen Qingbo Huineng Pharmaceutical Technology Co., Ltd. on October 18, 2019. Before the experiment, it was prepared into a 20 mM mother solution with DMSO and stored at -20 ° C. "Example 6" is a white powder stored dry at 4 ° C. and was supplied by Shenzhen Qingbo Huineng Pharmaceutical Technology Co., Ltd. on October 18, 2019. Before the experiment, it was prepared into a 20 mM mother solution with DMSO and stored at -20 ° C. Indomethacin, with lot number 1108939, was manufactured by Shanghai Crystallography Industry Co., Ltd. and supplied by Hangzhou Huante Biotechnology Co., Ltd. Before the experiment, it was dissolved in DMSO to prepare an 80 mM mother solution and stored at -20 °C.

[0260] Equipment and Reagents Dissecting microscope (SZX7, OLYMPUS, Japan), camera connected to the microscope (VertA1), precision electronic balance (CP214, OHAUS, AmericaCP214, OHAUS), fluorescence microscope (AZ100, Nikon, Japan), methylcellulose (Sigma, USA), dimethyl sulfoxide (Sigma, France), 6-well plate (Nest Biotech).

[0261] By concentration group 1. Determination of Minimum Toxic Concentration (MTC) for "Example 5" and "Example 6" Experimental group 1 Normal control group (no treatment) Experimental group 2 Model control group Experimental group 3 "Example 5" 0.625 μM Experimental group 4 "Example 5" 1.25 μM Experimental group 5 "Example 5" 2.5 μM Experimental group 6 "Example 5" 5 μM Experimental group 7 "Example 5" 10 μM Experimental group 8 "Example 5" 50 μM Experimental group 9 "Example 5" 100 μM Experimental group 10 "Example 5" 200 μM Experimental group 11 "Example 6" 0.625 μM Experimental group 12 "Example 6" 1.25 μM Experimental group 13 "Example 6" 2.5 μM Experimental group 14 "Example 6" 5 μM Experimental group 15 "Example 6" 10 μM Experimental group 16 "Example 6" 50 μM Experimental group 17 "Example 6" 100 μM Experimental group 18 "Example 6" 200 μM 2. Anti-inflammatory effect evaluation of "Example 5" and "Example 6" Experimental group 1 Normal control group (no treatment) Experimental group 2 Model control group Experimental group 3: Positive control drug indomethacin 80 μM Experimental group 4 "Example 5" 0.28 μM Experimental group 5 "Example 5" 0.83 μM Experimental group 6 "Example 5" 2.5 μM Experimental group 7 "Example 6" 0.28 μM Experimental group 8 "Example 6" 0.83 μM Experimental group 9 "Example 6" 2.5 μM

[0262] Concentration determination basis Based on the concentration search results and in consultation with the client, it was determined that the minimum toxic concentrations in the anti-inflammatory effect evaluation of "Example 5" and "Example 6" were both 2.5 μM.

[0263] Model creation We established a zebrafish inflammation model by treating normal 3 dpf transgenic neutrophil fluorescent zebrafish with LPS via intrayolk sac injection.

[0264] Experimental Method 1. Determination of Minimum Toxic Concentration (MTC) for "Example 5" and "Example 6" Healthy, stage-matched transgenic neutrophil fluorescent zebrafish at 3 days post-fertilization (dpf) were selected under a microscope and placed in a 6-well plate, with 30 randomly selected for each well, with a well volume of 3 mL, and LPS was injected into the yolk sac to establish a zebrafish inflammation model. "Example 5" and "Example 6" were dissolved in water and administered, with all concentrations of 0.625, 1.25, 2.5, 5, 10, 50, 100 and 200 μM. At the same time, a normal control group (i.e., zebrafish treated with culture water) and a model control group were set up. After being placed in a 28°C incubator and cultured for 3 hours, the death status of the zebrafish was observed and recorded, the number of zebrafish deaths in each experimental group was statistically recorded, and the minimum toxic concentration (MTC) of "Example 5" and "Example 6" for zebrafish was determined.

[0265] 2. Anti-inflammatory effect evaluation of "Example 5" and "Example 6" Healthy, stage-matched transgenic neutrophil fluorescent zebrafish at 3 days post-fertilization (dpf) were selected under a microscope and placed in a 6-well plate, 30 fish were randomly selected into each well, the volume of the well was 3 mL, and LPS was injected into the yolk sac to establish a zebrafish inflammation model. "Example 5" and "Example 6" were dissolved in water and administered, all at concentrations of 0.28, 0.83, and 2.5 μM. The indomethacin concentration was 80 μM. At the same time, a normal control group (i.e., zebrafish treated with culture water) and a model control group were set up. After being placed in a 28°C incubator and cultured for 3 hours, 10 zebrafish were randomly selected from each group, observed and photographed under a fluorescent microscope, and the images were saved. The images were analyzed using Nikon's NIS-Elements D 3.10 high-end image processing software, and the zebrafish inflammatory neutrophil count (N) was calculated. The statistical processing results were shown as mean ± SE. The statistical analysis results of the zebrafish inflammatory neutrophil count were used to evaluate the significance of the anti-inflammatory effects of "Example 5" and "Example 6" on LPS-induced inflammatory zebrafish, respectively. The calculation formulas for the anti-inflammatory effects of "Example 5" and "Example 6" are as follows: Inflammation reduction effect (%) = ((N (model control group) - N (test product group)) / N (model control group)) * 100% Statistical analysis was performed by analysis of variance and Dunnett's T-test, with significant differences indicated at p < 0.05.

[0266] Experimental Results 1. MTC Since the maximum solubility of "Example 5" in DMSO is 20 mM and the maximum DMSO concentration that zebrafish can tolerate is 1%, the minimum toxic concentration for evaluating the anti-inflammatory effect of "Example 5" is 200 μM. At the 200, 100, 50 and 10 μM concentrations of "Example 5", 30 zebrafish died, with a mortality rate of 100%. At the 5 μM concentration, 3 zebrafish died, with a mortality rate of 10%. At the 2.5 μM concentration, the zebrafish condition was normal and the drug did not precipitate. The minimum toxic concentration for evaluating the anti-inflammatory effect of "Example 5" was 2.5 μM. Since the maximum solubility of "Example 6" in DMSO is 20 mM and the maximum DMSO concentration that zebrafish can tolerate is 1%, the maximum administration concentration for evaluating the anti-inflammatory effect of "Example 6" is 200 μM. At the 200, 100, 50 and 50 μM concentrations of "Example 6", 30 zebrafish died, with a mortality rate of 100%. At the 10 and 5 μM concentrations, 4 zebrafish died, with a mortality rate of 13.33%. At the 2.5 μM concentration, the zebrafish condition was normal and the drug did not precipitate. The minimum toxic concentration for evaluating the anti-inflammatory effect of "Example 6" was 2.5 μM. The minimum toxic concentrations of "Example 5" and "Example 6" were both in accordance with the concentration search results, that is, the minimum toxic concentrations for evaluating the anti-inflammatory effects of "Example 5" and "Example 6" were both 2.5 μM. See Table 3 for details.

[0267] [Table 3]

[0268] 1.Anti-inflammatory effect As shown in Table 4, Figure 4, Figure 5, and Figure 6, the dashed area in Figure 4 indicates neutrophils at the inflammatory site. Comparing the neutrophil counts at the inflammatory site of the model control group zebrafish (18 cells) with the normal control group (3 cells), p < 0.001, indicates that the LPS-induced inflammation model of transgenic neutrophil fluorescent zebrafish was successfully established. Comparing the neutrophil counts at the inflammatory site of the 80 μM indomethacin group zebrafish (6 cells) with the model control group, p < 0.001, the anti-inflammatory effect was 67%, indicating that indomethacin has a clear anti-inflammatory effect on inflamed zebrafish.

[0269] When the concentrations of "Example 5" were 0.28, 0.83, and 2.5 μM, the neutrophil counts at the inflamed 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 0.28, 0.83, and 2.5 μM concentration groups all had p < 0.001, indicating that "Compound 3" had a clear anti-inflammatory effect on inflamed zebrafish at concentrations of 0.28 to 2.5 μM.

[0270] When the concentrations of "Example 6" were 0.28, 0.83, and 2.5 μM, the neutrophil counts at the inflamed site of the zebrafish were 12, 8, and 7, respectively, and the anti-inflammatory effect on the zebrafish was 33%, 56%, and 61%, respectively. Compared with the model control group (18), the 0.28, 0.83, and 2.5 μM concentration groups all had p < 0.001, indicating that "Compound 4" had a clear anti-inflammatory effect on the inflamed zebrafish when the concentration was 0.28 to 2.5 μM.

[0271] [Table 4]

[0272] Experimental Conclusion Under the concentration conditions of this experiment, both Example 5 and Example 6 exhibited a clear anti-inflammatory effect on inflamed zebrafish. Experiment 5: Pharmaceutical study to evaluate the effect of compounds on mouse cerebral ischemia-reperfusion (MCAO) injury model The effects of the compounds on mouse cerebral ischemia-reperfusion injury (MCAO) model were evaluated using neurological function scores and cerebral infarct size, and the results showed that the test compounds had protective effects on mouse cerebral ischemia-reperfusion injury (MCAO).

[0273] Experimental Agents [Table 5]

[0274] Saline solution Name: In-house produced saline solution. Hydroxypropyl-β-cyclodextrin Name: Hydroxypropyl-β-cyclodextrin, Supplier: Saen Chemical Technology (Shanghai) Co., Ltd., Lot number: FG310174, Properties: White solid powder, Quantity: 30g, Storage conditions: Room temperature.

[0275] Experimental methods Experimental animals and breeding Experimental animals: strain: C57BL / 6 mouse, age: 6-8 weeks, sex: male, purchased animal weight: 16-20 g, used animal weight: 20-23 g, quantity: 40, experimental animal provider: Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., production permit number: SCKX(Zhejiang)2019-0001, quality certification number: No2005130052, No2004280010.

[0276] Animal husbandry Quarantine: The quarantine period was 7 days, during which a veterinarian performed routine health checks, and any animals exhibiting abnormal behavior were removed before the experiment. Animal breeding conditions: Experimental animals were kept in a laminar flow clean room with constant temperature and humidity of SPF class in the animal center (AAALAC certified), with 3 animals per cage. The temperature of the breeding room was 22 ± 3℃, the humidity was 40-70%, and the lighting was alternated between light and dark every 12 hours. Cages: Made of polycarbonate. The bedding was clean, autoclaved, and soft corn cob bedding was changed twice a week. Food and drinking water: Clean-class mouse food purchased from Beijing Keao Cooperative Feed Co., Ltd. The drinking water was sterilized by autoclave, and the food was irradiated with cobalt 60. The animals had free access to sterile food and drinking water. Animal Numbering: Each cage was labeled with the animal number, sex, breed, time of receipt, group and start time of the experiment. Animal Numbering: Each animal was given an individual animal number on its tail. Animal grouping and treatment: According to the animal weight, the animals were randomly divided into 8 groups on the first day. N=5, 8 groups per system, one group was experimented per day, and 3 animals were selected from each group to measure cerebral blood flow. For specific grouping and treatment, see Table 6.

[0277] [Table 6]

[0278] MCAO model preparation Mice were fasted overnight before surgery, but were allowed to drink water. Mice were preanesthetized with an isoflurane gas anesthesia induction box at a concentration of 2.5%. After the mice were held with dissection forceps and showed no reaction in their hind limbs, they were transferred to an anesthesia mask and the isoflurane concentration was reduced to 1.5%. The body temperature of the mice was maintained at around 37°C during surgery using a body temperature maintenance device and an anal temperature probe. The neck of the mice was shaved in a supine position, and the skin was disinfected with iodophor and alcohol, after which a central neck incision was made. The tissue was bluntly dissected to expose the common carotid artery (CCA) under a stereomicroscope, and its proximal end was ligated with 6-0 braided wire. The external carotid artery (ECA) and internal carotid artery (ICA) were separated upward, and a permanent ligation was performed at the distal end of the ECA, a temporary ligation was performed at the distal end of the ECA, and a temporary ligation was performed at the carotid sinus. The ECA wall was cut and a suture plug (Beijing Xinong Technology Co., Ltd., A5-162020) was inserted, and the temporary ligation was pulled tight to fix the suture plug. The ECA was fused with an electrocoagulation pen, the ligation of the external carotid artery was loosened, and a suture plug was inserted into the ICA until cerebral blood flow was approximately 10% of baseline. The middle cerebral artery (MCA) was occluded with a suture plug for 30 min, after which the suture plug was removed, the vascular remnant was cauterized, and the CCA ligation was loosened. The skin of the neck was sutured, and the postoperative body temperature was maintained at 37°C until specimen collection, and the mouse was placed in an intensive care cage.

[0279] Measurement of cerebral blood flow: The mouse head was fixed under a stereotaxic apparatus, and after shaving, a midline incision was made. The periosteum was removed from the skull, and the optical fiber of the laser Doppler blood flow meter was fixed with adhesive at the coordinates of Bregma AP 1.0 mm, ML 5.0 mm, and intraoperative blood flow changes in the middle cerebral artery were recorded. The success criterion for modeling was a reduction in cerebral blood flow to 80-90% of baseline.

[0280] Drug preparation Solvent: Saline Solvent: 20% hydroxypropyl-β-cyclodextrin PK sample preparation: The minimum daily dose m2 was calculated based on the total weight of each group of mice and the drug dose. A slightly larger amount of P2 than m2 was weighed into a bottle, and the solvent volume v2 was calculated based on the administration volume of 10 mL / kg. 20% hydroxypropyl-β-cyclodextrin solution (v2) was added to the bottle, vortexed for 1 minute, sonicated for 15 minutes, and vortexed for another minute until the solution became clear. Solvent: 20% hydroxypropyl-β-cyclodextrin PK sample preparation: The minimum daily dose m2 was calculated based on the total weight of the mice in each group and the drug dose. A slightly larger amount of P3 than m3 was weighed into a mortar, and the solvent volume v3 was calculated based on the dose volume of 10 mL / kg. The solvent v2 was measured with a syringe. 2-3 drops of solvent were added to the mortar, ground for 5 minutes, and after the solvent dried, another 2-3 drops of solvent were added and ground for 5 minutes, and this was repeated three times. The compound in the mortar was then washed multiple times with small amounts of solvent, and added to an appropriate vial using a clean glass dropper, sonicated for 15 minutes until no large particles were present and the mixture was homogenous, and vortexed for 5 minutes.

[0281] Metrics and methods Body weight: The body weight of the mice was recorded on the day before surgery and 24 h after surgery. Neurofunctional score: Mice were subjected to Longa behavioral scoring 2 h and 24 h after surgery. Longa Behavioral Scoring Criteria: 0 points: no symptoms of nerve damage; 1 point: Complete inability to extend the contralateral forepaw; 2 points: Turn to the other side; 3 points: Fall to the opposite side; 4 points: Inability to walk spontaneously, loss of consciousness.

[0282] Forelimb grip strength measurement The grip strength of the mouse forelimbs was measured 24 hours before and after surgery. The mouse was placed on a grip strength meter and actively pulled the grip strength sensing lever. The peak grip strength was recorded and the measurement was repeated three times to calculate the average value.

[0283] infarct area 24 hours after surgery, mice were anesthetized with isoflurane and then decapitated. The whole brain was removed and washed twice with saline, then placed under a coronal brain mold, and the olfactory bulb 1 mm anterior and the cerebellum 4 mm posterior were removed. The brain was cut into 2 mm slices, for a total of four slices. Brain slices were incubated and stained in 1% TTC solution at 37°C in the dark, then stored in 4% PFA at 4°C for 48 hours, and then photographed. Cerebral infarction volume and edema volume were measured using ImageJ software. Brain edema volume = (volume of the injured hemibrain - volume of the contralateral hemibrain) / volume of the contralateral hemibrain * 100% Corrected cerebral infarction volume = (contralateral hemibrain volume - (damaged hemibrain volume - white infarct volume)) / contralateral hemibrain volume * 100%

[0284] statistics The data of each group was statistically analyzed using Graghpad Prism 7.0, and the experimental results were expressed as "mean ± standard deviation". One-way analysis of variance was used to compare whether there was a statistical difference between each group, and P<0.05 was considered to be a statistical difference.

[0285] Experimental Results As shown in Figure 7, MCAO surgery reduced cerebral blood flow in each group (n=3) to approximately 10% of baseline on average, indicating successful modeling and enabling pharmacological evaluation. As shown in FIG. 8, after surgery, the body weight of each group decreased, but no significant difference was observed in the body weight of mice in each group that underwent MCAO surgery. As shown in Figure 9, the forelimb grip strength of mice in each group was obviously decreased after surgery, and among them, significant differences were observed between the sham operation group and the MCAO model group (P < 0.001), between the MCAO model group and the positive control drug edaravone group (P < 0.001), and between the MCAO model group and the No. 3 administration group (P < 0.001). As shown in Figure 10, 2 hours after surgery, the nerve function deficit score of the MCAO model group was significantly different from that of the No. 2 and No. 3 groups (P < 0.05), and 24 hours after surgery, the nerve function deficit score of the MCAO model group was significantly different from that of the positive control drug edaravone group and No. 3 group (P < 0.05). As shown in Figure 11, the TTC staining results showed significant differences between the cerebral infarction volume of the MCAO model group and the positive control drug edaravone group (P < 0.001), No. 2 (P < 0.001), No. 3 (P < 0.001), No. 4 (P < 0.01) and No. 5 drug (P < 0.01). As shown in Figure 12, the TTC staining results showed significant differences in the brain edema volume between the MCAO model group and the positive control drug edaravone group (P < 0.01), No. 2 (P < 0.001), No. 3 (P < 0.001), No. 4 (P < 0.001), No. 5 drug (P < 0.05) and No. 6 drug (P < 0.05).

[0286] Experiment 6: Evaluation of the effect of Example 6 on LPS-induced depression-like mouse model LPS was used to induce peripheral and central inflammation in mice, induce depression-like symptoms in the mice, and detect changes in depression-related behavioral indicators in the mice after drug administration. As a result, it was shown that Example 6 (ND) has an antidepressant effect in mice.

[0287] [Table 7] Solvent: Carmellose sodium (NC), Name: Carmellose sodium, Supplier: Miner, Lot number: 69881020, Properties: White solid powder, Quantity: 200g, Storage conditions: Room temperature Peripheral and central inflammatory inducer (LPS) Name: Lipopolysaccharide, Manufacturer: Sigma, Lot number: L2880-100MG, Properties: White powder, Purity: >97%, Quantity: 100mg, Storage conditions: 2-8℃, Protected from light

[0288] Experimental methods Experimental animals and breeding management Experimental animal strain: C57BL / 6J mouse, age: 6-8 weeks, sex: male, animal weight: 18-20 g, quantity: 24, experimental animal provider: Guangdong Medical Experimental Animal Center

[0289] Animal husbandry Quarantine: The quarantine period was 5 days, and animals showing abnormal behavior were removed before the experiment. Animal breeding conditions: Experimental animals were kept in a clean-class laminar flow clean room with constant temperature and humidity at the Animal Center of the Shenzhen Research Institute of Peking University, with 4-5 animals per cage. The temperature of the breeding room was 22 ± 3°C, the humidity was 40-70%, and the lighting was alternated between light and dark every 12 hours. Food and drinking water: clean-class mouse food purchased from Beijing Keao Cooperative Feed Co., Ltd. Drinking water was sterilized by autoclave. The animals were allowed free access to sterile food and drinking water. Animal Numbering: Each cage was labeled with the animal number, sex, breed, time of receipt, group and start time of the experiment. Animal Numbering: Each animal was given an individual animal number on its tail. Animal grouping and treatment: Animals were randomly divided into 3 groups, see Table 8 for specific groupings and treatments.

[0290] [Table 8]

[0291] Establishment of an LPS-induced depression-like model Modeling method: LPS (2mg / kg,ip,QD) was injected to induce peripheral and central inflammation in mice on days 1, 2, and 3. The ND drug test group was temporarily protected by drug administration (ND 30mg / kg,po,QD) 2 hours before LPS injection, and forced swimming, tail suspension, and sucrose preference tests were performed on D4.

[0292] Preparation of test agents and model inducers Solvent (0.5% CMC-Na solution): Take 0.5g of CMC-Na white solid powder, add 100 mL of double distilled water, and vortex until it dissolves transparently. The prepared solution was placed in a refrigerator at 4°C, sealed, and stored. The solution can be stored for one month, and if mold is found, do not use it in the experiment and prepare it again. During the experiment, the test compounds and model inducers were prepared immediately before use. The specific preparation method is shown in Table 9.

[0293] [Table 9]

[0294] Metrics and methods General Observation: The living conditions of the animals were observed and recorded. Forced swim test (FST) Each mouse was placed in a transparent glass cylinder (diameter: 23 cm, height: 31 cm), and 15 cm of water was added, with the temperature maintained at 24 ± 1 ° C. The FST lasted for 6 minutes, during which time the mice were photographed with a high-resolution camera and the immobility time during the test was measured with professional measurement software. After completion, the mice were immediately returned to their cages and kept warm. Sucrose preference test (SPT) Before the test, the mice were trained; that is, two bottles of 1% (W / V) sucrose solution were placed in each cage, and one of them was replaced with pure water after 24 hours. After adaptation, the test was performed after 10-24 hours of fasting. Two pre-weighed bottles were placed in each cage, one with 1% (W / V) sucrose solution and the other with pure water. After 12 hours, one bottle was weighed again, and the consumption of sucrose solution and pure water for each mouse was recorded. Sucrose orientation index %=sucrose solution consumption / (sucrose solution volume+pure water consumption)×100%. Tail suspension test (TST) After adapting to the environment, the mouse's head was placed about 20-25 cm above the ground, and the mouse's tail was attached to a hanging bar for about 6 minutes. The mouse was photographed with a high-resolution camera, and the mouse's immobility time was identified and statistically recorded using behavioral analysis software. After the experiment was completed, the mouse was returned to its cage. statistics The experimental results were expressed as the mean ± standard deviation. Data from each group were compared for statistical differences between groups using one-way ANOVA.

[0295] Experimental Results FST As shown in FIG. 13, the results of FST immobility time for each mouse group showed that the cumulative immobility time for Example 6 (ND) was significantly lower than that for the LPS group. **, P<0.01; ***, P<0.001, SPT As shown in Figure 14, the sucrose preference results of the mouse SPTs in each group were not significantly higher than those of the Example 6 (ND) and LPS groups. *, P<0.05; ***, P<0.001 TST As shown in Figure 15, the results of mouse TST immobility time for each group showed that the cumulative immobility time in Example 6 (ND) was significantly lower than that in the LPS group. **, P<0.05; ***, P<0.001 Conclusion: Compound Example 6 (ND) showed good antidepressant effects in FST and TST experiments.

[0296] Experiment 7. Evaluation of the effect of compound Example 20A on LPS-induced peripheral and central inflammation models in mice Peripheral and central inflammation was induced in mice by LPS induction, and changes in inflammation and oxidative stress levels in serum and hippocampal tissue of mice after drug administration were measured. As a result, Example 20A (ND) showed a clear anti-inflammatory effect. Sample [Table 10] solvent Carmellose sodium (NC), Name: Carmellose sodium, Provider: Miner, Lot number: 69881020, Properties: White solid powder, Quantity: 200g, Storage conditions: Room temperature. Peripheral and central inflammatory inducer (LPS), name: lipopolysaccharide, manufacturer: Sigma, lot number: L2880-100MG, properties: white powder, purity: >97%, amount: 100mg, storage conditions: 2-8℃, protected from light.

[0297] Experimental Method Laboratory animals and husbandry Experimental animal strain: C57BL / 6J mouse, age: 6-8 weeks, sex: male, animal weight: 18-20 g, quantity: 24. Experimental animal provider: Guangdong Provincial Medical Experimental Animal Center Animal husbandry Quarantine: The quarantine period was 5 days, and animals showing abnormal behavior were removed before the experiment. Animal breeding conditions: Experimental animals were kept in a clean-class laminar flow clean room with constant temperature and humidity at the Animal Center of the Shenzhen Research Institute of Peking University, with 4-5 animals per cage. The temperature of the breeding room was 22 ± 3°C, the humidity was 40-70%, and the lighting was alternated between light and dark every 12 hours. Food and drinking water: clean-class mouse food purchased from Beijing Keao Cooperative Feed Co., Ltd. Drinking water was sterilized by autoclave. The animals were allowed free access to sterile food and drinking water. Animal Numbering: Each cage was labeled with the animal number, sex, breed, time of receipt, group and start time of the experiment. Animal Numbering: Each animal was given an individual animal number on its tail.

[0298] Animal grouping and treatment Animals were randomly divided into 3 groups, see Table 11 for specific groupings and treatments. [Table 11]

[0299] Establishment of LPS inflammation model Modeling method: On days 1, 2, and 3, mice were injected with LPS (2mg / kg,ip,QD) to induce peripheral and central inflammation. The ND drug test group was temporarily protected by drug administration (ND 30mg / kg,po,QD) 2 hours before LPS injection. After the experiment was completed, mouse plasma and brain tissue were collected to measure indicators such as inflammatory and peroxidative factors.

[0300] Preparation of test agents and model inducers Solvent (0.5% CMC-Na solution): Take 0.5g of CMC-Na white solid powder, add 100 mL of double distilled water, and vortex until it dissolves transparently. The prepared solution was placed in a refrigerator at 4°C, sealed, and stored. The solution can be stored for one month, and if mold is found, do not use it in the experiment and prepare it again.

[0301] During the experiment, the test compounds and model inducers were prepared immediately before use. The specific preparation method is shown in Table 12. [Table 12]

[0302] Metrics and methods General Observation: The living conditions of the animals were observed and recorded.

[0303] Animal specimen collection Serum: After anesthetizing the mice with 1% sodium pentobarbital, the eyeballs were enucleated and blood was collected into EP tubes. The blood was then left to stand at 4°C for 1 hour, centrifuged at low temperature, and the supernatant was removed and quickly frozen in liquid nitrogen. Hippocampal tissue: Mice were anesthetized with 1% sodium pentobarbital, then cardiac perfusion was performed with cold PBS, and the brain was removed. The tissue was then collected from ice into an EP tube and quickly frozen in liquid nitrogen.

[0304] Measurement of oxidative stress-related indicators Frozen plasma was collected and each indicator was detected using an active oxygen detection kit, a hydrogen peroxide detection kit, and a nitric oxide detection kit. Specific operating procedures were performed according to the instructions for each kit. Frozen hippocampal tissue was collected, homogenized thoroughly with RIPA tissue lysis solution containing a protease inhibitor, and dissolved. After low-temperature centrifugation, the supernatant was collected and protein was quantified using a BCA protein quantification kit. The protein concentration of each tissue sample was standardized, and each indicator was measured using an active oxygen detection kit, a hydrogen peroxide detection kit, and a nitric oxide detection kit. The specific operating procedures were according to the instructions for each kit.

[0305] Cytokine measurements Frozen plasma was collected and each indicator was measured using IL-1β, IL-6, and IL-10 kits, with specific operating procedures following the instructions for each kit. The supernatant of the early hippocampal tissue was taken, and each indicator was measured using IL-1β, IL-6, and IL-10 kits. The specific operating procedures were according to the instructions for each kit.

[0306] statistics The experimental results were expressed as the mean ± standard deviation. Data from each group were compared for statistical differences between groups using one-way ANOVA.

[0307] Experimental Results Oxidative stress-related indicators As shown in Figures 16 to 23, the serum and hippocampal tissue oxidative stress indexes of mice in each group were measured, and the ROS level, H2O2 concentration, and NO concentration in the serum of Example 20A (ND) were significantly improved compared to the LPS group, indicating that Example 20A (ND) had good antioxidant effects. *, P<0.05; **, P<0.01. Inflammatory factor index As shown in Figures 24 to 29, the serum and hippocampal tissue inflammation-related important indicators of mice in each group were measured, and it was shown that Example 20A only affected IL-1β in serum and IL-10 in hippocampus, and Example 20A (ND) had anti-inflammatory effects. *, P<0.05; **, P<0.01.

[0308] Experiment 8: Measurement of the effect of small molecule compounds on the binding strength of mitochondrial respiratory chain supercomplexes Experimental and analytical methods: HEK293 cells were cultured under 5% CO2 conditions, and the mitochondrial respiratory chain supercomplex I1III2IV1 was purified from the cells. After the respiratory chain supercomplex was chemically covalently bound to the surface of a metal chip, different concentrations of small molecule compounds were passed through the metal chip, and the change in the metal surface reflectance coefficient was monitored using a Biacore 8K plus analyzer, and a response curve corresponding to small molecule binding was plotted. The binding strength (KD value) between the small molecule and human-derived mitochondrial respiratory chain supercomplex I1III2IV1 was calculated using the fitting software built into the Biacore 8K plus instrument. Compound treatment: The small molecules shown in the figure were dissolved in ddH2O to prepare a 10 mM mixed stock solution. Next, the mixed stock solution gradient of Example 13B was diluted to 1.5625 μM, 0.78125 μM, 0.390625 μM, and 0.1953125 μM with 10 mM HEPES (pH = 7.4) solution. Similarly, the mixed stock solution gradient of Example 1A was diluted to 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, 0.390625 μM, and 0.1953125 μM with 10 mM HEPES (pH = 7.4) solution. At the time of measurement, 200 μL of the small molecule solution of each concentration was taken and passed over the metal chip surface. The results are shown in Figures 30a to 32b, and the binding strengths of the small molecules to respiratory chain complex I all reached high levels.

[0309] Experiment 9. Measurement of SMP (submitochondrial particle) catalytic activity by small molecule compounds Experimental and analytical methods: HEK293 cells were cultured under 5% CO2 conditions, mitochondria were purified from them, and the mitochondria were sonicated to obtain SMP (submitochondrial particles). SMP was incubated with different concentrations of small molecule compounds or a control drug (Rotenone, 2 μM) for 10 minutes, and then 500 μM NADH was added. The absorbance of NADH was measured using an Enspire multimode plate reader, and the enzyme activity curve of the NADH concentration reduction catalyzed by SMP was plotted and linear fitting was performed to calculate the maximum reaction rate of SMP. FeCN 1 mM was added to the normal NADH catalytic system of SMP, allowing the electron pathway from NADH to coenzyme Q to be short-circuited. In the presence of FeCN and Rotenone, the NADH concentration reduction curve was similarly measured, linear fitting was performed, and the maximum reaction rate of the SMP flavin yellow site was calculated. Compound treatment: The small molecules shown in the figure were dissolved in ddHO to prepare a 10 mM mixed stock solution, and a small molecule gradient was diluted with 10 mM Tris buffer solution to 0.075, 0.1, 0.133, 0.178, 0.237, 0.316, 0.422, 0.563, 0.75, and 1 μM to perform SMP catalysis experiments. As a result, as shown in Figure 33, in the catalytic experiment system, the control group had catalytic activity, and rotenone completely blocked the dehydrogenation reaction of NADH, indicating that the reaction system was effective. Small molecules within 1 μM shown in the figure had no significant effect on the activity of SMP. On the other hand, small molecules at high concentrations shown in the figure had a large effect on the activity of SMP.

[0310] Experiment 10. Measurement of HPAEC cell mitochondrial stress promotion by small molecule compounds. Experimental and analytical methods: HPAEC cells (human pulmonary artery endothelial cells) were purchased from ATCC and subcultured four times under 5% CO2 culture conditions, then transferred to a Seahorse dedicated cell plate containing approximately 20,000 cells per well. After the cells attached to the wall, 0.5, 1 μM of Example 13B and HPAEC cells were cultured for 48 hours, and then the oxygen consumption rate (OCR) of each well cell was measured using a Seahorse XFe96 analyzer, and the OCR curve was plotted. After the measurement was completed, the total protein content in each cell well was measured using BCA, and the OCR curve was corrected. The results showed that, as shown in Figure 34, in the resting state (time: 0-20 min), the five compounds had little effect on the oxygen consumption of the cells. After adding oligomycin, an inhibitor of complex V, the proton gradient was not consumed, and although a high proton gradient inhibits the activity of complex I and leads to a decrease in the oxygen consumption rate, there was little difference between different small molecules and the control group. When the uncoupler FCCP was added, the proton gradient dropped sharply, the oxygen consumption rate increased dramatically, and all small molecules accelerated the oxygen consumption rate. This reflected that these small molecules increased the sensitivity of the reaction of complex I to the sudden drop in the proton gradient, and complex I could promote a stronger increase in the electron transport rate. Finally, when rotenone (a mitochondrial electron transport chain complex I inhibitor) and antimycin (a mitochondrial electron transport chain complex III inhibitor) were added, the electron transport was completely blocked, the oxygen consumption rate was reduced to the limit, and no effect of small molecules on the blockage of electron transport was observed.

[0311] Experiment 11. Measurement of small molecule compounds that protect HPAEC cells from ROS production induced by the novel coronavirus spike. Experimental and analytical methods: HPAEC cells (human pulmonary artery endothelial cells) were purchased from ATCC and subcultured four times under 5% CO2 culture conditions, then transferred to a MatTek cell imaging dish at approximately 50,000 cells per slot. HPAEC cells were cultured with spike (new coronavirus surface spike protein, 8 μg / mL) for 24 hours, or with Example 13B (0.25, 0.5, 1 μM) for 6 hours, and then cultured with spike (8 μg / mL) for 24 hours. Next, mitochondria were stained with MitoSox Red, PKMDR, and Mitotracker Green mitochondrial fluorescent probes, and confocal fluorescent photographs of the cells were taken with a Leica stimulated emission depletion super-resolution confocal fluorescence microscope (STED). Then, the cell fluorescence intensity in each group was measured using ImageJ, and the standard deviation and standard error were calculated from the obtained data, and the P value was calculated by T-test analysis to determine whether there was a significant difference. Results: After spiking into HPAEC cells, mitochondrial ROS increased significantly (red fluorescence became brighter) compared to the control group. As shown in Figures 35a-35b, mitochondrial ROS was obviously reduced when mitochondrial protection was followed by spiking with the small molecules shown in the figures. When the PKMDR / MTG value is used as an index to evaluate the mitochondrial transmembrane potential per unit mass, the small molecules shown in the figures were shown to have a protective effect on the mitochondrial transmembrane potential.

[0312] Experiment 12. Measurement of atherosclerosis improvement in APOE mice by small molecules Experimental and analytical methods: After purchasing 6-week-old male APOE- / - mutant B6J mice and WT B6J mice, they were adapted for 2 weeks in the breeding room. When the mice were 8 weeks old, the groups except the vehicle group and the WT group were administered a high-fat diet (Research Diets, D12492). When the mice were 9 weeks old, the control group was administered a 10% cyclodextrin solution, and the other treatment groups began to receive different concentrations of small molecule compounds orally and intragastrically once every 2 days. At 1 month of administration, approximately 500 μL of blood was collected from the orbital venous plexus of the APOE mice, centrifuged, and the supernatant plasma was aspirated, and the total cholesterol (CHOL), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels in the plasma were measured. After 16 weeks of administration of the high-fat diet, the APOE mice were sacrificed and the aortic arches were dissected and collected. After fixing the aortic arch of the mouse with 4% polyacetal, the arterial plaque inside the aortic arch was stained with oil red, and the stained aortic arch was imaged with a Zeiss stereomicroscope. Using the photographs taken, the total area of ​​the aortic arch longitudinal section and the area of ​​orange-red plaque were analyzed using ImageJ, and the percentage of plaque area was calculated. The data was analyzed by T-test to calculate the P value and determine whether there was a significant difference. Compound treatment: The indicated small molecules (Figures 36a-36d) were dissolved in 10% cyclodextrin solution to a final concentration of 12 mg / mL, then diluted in gradients to 6, 3, and 1.5 mg / mL in 10% cyclodextrin solution. The results are shown in Figures 36a-36d: after administration of a high-fat diet, the total cholesterol level in the serum of the APOE mice in the control group reached about 800 mg / dL, whereas the total cholesterol in the serum of the WT genotype mice was only about 100 mg / dL. After administration of the small molecules shown in the figures, the total cholesterol levels of the APOE mice were all reduced. In addition, the small molecules shown in the figures have little effect on the high-density lipoproteins of the APOE mice, but the trend of the low-density lipoproteins is almost the same as that of the total cholesterol, indicating that the small molecules shown in the figures mainly reduced bad LDL but maintained good HDL. As can be seen from the oil red staining of the aortic arch of APOE, the treatment group had much less orange-red plaque area than the control group, indicating a statistically significant difference. The above results indicate that the small molecules shown in the figures can suppress the development of atherosclerosis by reducing the LDL level in serum.

[0313] Experiment 13. AD rat water maze Experimental and analytical methods: A transgenic AD rat model was obtained by introducing human APP mutant protein into SD rat embryos using CRISPR Cas9 technology. This strain of rats began to have amyloid deposits in the brain from the age of 2 months, and began to show some degree of behavioral impairment at the age of 5 months. To verify the therapeutic effect, the rats were administered from the age of 7 to 13 months, and water maze experiments were performed to verify the improvement of their spatial memory. The water maze experiment process was divided into three stages: the first stage was the adaptation stage, which lasted for 1 day, and was a large 1.8m diameter pool with a 15cm diameter escape platform. As a hint for spatial location, four marks were placed on the pool wall in the east-south-west-north directions, namely a circle, a triangle, a square, and a cross. The rats were sequentially placed in the transparent water along the pool wall, and the water surface was 1cm below the escape platform. The rats easily found the escape platform, climbed onto the platform, and were rescued from the water maze, thereby acquiring the basic rules of the water maze. The second stage was the training stage, which lasted for 4 days. The position of the pool was not changed, but the water surface was set at 1 cm above the escape platform, and ink was added to the water to dye it black so that the escape platform could not be seen. Every day, rats were placed in the pool from four directions (east, south, west, north) and rescued after climbing onto the escape platform. If the rat could not find the platform within 90 s, it was artificially induced to climb onto the platform, and was rescued after standing on the platform for 15 s. The third phase lasted for 1 day and was the test phase. The rat was removed from the escape platform and placed again along the pool wall. Rats with strong spatial memory repeatedly made round trips to the original escape platform position. Indices such as the latency to first arrive at the escape platform, the number of round trips to the escape platform, the average distance to the escape platform, and the residence time in the target quadrant were recorded. Results: There were three groups: 13 rats in the WT group, 7 rats in the model group (AD group), and 11 rats in the treatment group (Example 13B group). The behavioral performance of the treatment group was improved to some extent compared to the model group in terms of indicators such as the latency to first arrive at the escape platform (shown in Figure 37a), the number of trips to the escape platform (shown in Figure 37b), the average distance to the escape platform (shown in Figure 37c), and the residence time in the target quadrant (shown in Figure 37d). Figures 37e to 37g show the movement trajectory heat maps in the pool for rats in each group.

[0314] Experiment 14. AD rat T-maze Experimental and analytical methods: A transgenic AD rat model was obtained by introducing human APP mutant proteins into SD rat embryos using CRISPR Cas9 technology. This strain of rats began to have amyloid deposits in the brain from the age of 2 months, and began to show some degree of behavioral impairment at the age of 5 months. To verify the therapeutic effect, rats were administered from 7 to 15 months of age, and a T-maze experiment was performed to verify the improvement of their working memory. The T-maze was divided into four stages: I. Adaptation stage, both arms were filled with food, and both arms' valves were open. Each rat was grouped twice. If all rats finished eating both sides of the food within 5 minutes both times, they proceeded to the next stage. II. Forced choice training stage: The bubble on one side was closed, and the rat was allowed to eat the food on the other side. Next, the valve on the other side was closed, and the rat was allowed to eat the food on the other side. This was considered as one training session. Each rat was trained 4 times / day for 3 days. II. Self-choice training stage: The bubble on one side was closed, and the rat was allowed to eat the food on the other side. Then, all valves were opened, and if the rat moved to the other side, it could finish eating the food on the other side. If the rat moved to the same side, there was no food and it was confined on that side for 30s. This was considered as one training session. Each rat continued training four times / day, and when the accuracy rate exceeded 75% for two consecutive days, it moved on to the next stage. IV. Delayed self-choice stage: The operation was almost the same as in stage III, except that an interval was inserted between the completion of the forced choice and the start of self-choice. The interval times were set to 1.5 min, 3 min, and 10 min. Four tests were conducted for each interval time. Results: There were three groups of rats: WT group (3 rats), model group (AD group) (2 rats), and administration group (Example 13B group) (6 rats). As shown in Figure 38, at 1.5 min and 3 min, there was no obvious difference between the model group and the administration group, but at 10 min, the working memory of the model group was obviously reduced, while the working memory of the administration group still existed.

[0315] Experiment 15. Nest-building behavior in AD mice Experimental and analytical methods: The transgenic mice used in this experiment were transgenic mice introduced with APP / P Example 13B, and the background mice were C57BL / 6J. The phenotype of the model mice was that they had cognitive behavioral changes at 3 months of age, senile lentigo at 5 months of age, and a large number of senile lentigo at 12 months of age. One of the symptoms of AD patients is the inability to take care of themselves, and nest-building behavior reflects the self-care ability of mice. At the start of the experiment, 10 rectangular pieces of paper were neatly arranged in the mouse cage. Normal mice shred the paper to make nests, but mice with severe AD symptoms did not shred the paper to make nests. The degree to which the paper was shredded and the completeness of the nest-building were scored, and the higher the score, the better the nest-building behavior of the mice and the higher their self-care ability. Results: In this experiment, administration was started from 2 months of age, and nest-building behavior was scored 3 months and 6 months after administration. Four small molecules, Example 23, Example 24, Example 25, and Example 26, were tested. The results are shown in Figures 39a-39b. Compared with the WT group, the nest-building behavior of the model group was obviously weakened, and the improvement of the nest-building behavior of Example 25 was the most obvious.

[0316] Experiment 16. AD Mouse Water Maze Experimental and analytical methods: The transgenic mice used in this experiment were transgenic mice introduced with APP / P Example 13B, and the background mouse was C57BL / 6J. The phenotype of this model mouse was cognitive behavioral changes at 3 months of age, senile pigmentation at 5 months of age, and formation of a large number of senile pigmentation at 12 months of age. The water maze experiment process was divided into three stages: the first stage was the adaptation stage lasting 1 day, and included an escape platform with a diameter of 8 cm in a large pool with a diameter of 1.2 m. As a hint for spatial location, four marks were placed on the pool wall in the east-south-west-north directions, namely a circle, a triangle, a square, and a cross. The rats were sequentially placed in the transparent water along the pool wall, and the water surface was 1 cm below the escape platform. The mice easily found the escape platform, climbed onto the platform, and were rescued from the water maze, thereby learning the basic rules of the water maze. The second stage was the training stage lasting 4 days. The position of the pool was not changed, but the water surface was set at 1 cm above the escape platform, and ink was added to the water to dye it black so that the escape platform could not be seen. Every day, mice were placed in the pool from four directions (east, south, west, north) and rescued after climbing onto the escape platform. If the rat could not find the platform within 90 s, it was artificially induced to climb onto the platform, and was rescued after standing on the platform for 15 s. The third stage lasted for 1 day and was the test stage. The rat was removed from the escape platform and placed again along the pool wall. Rats with strong spatial memory repeatedly returned to the original escape platform position. Indicators such as the number of times the rat returned to the escape platform and the time spent in the target quadrant were recorded. Results: In this experiment, the mice were administered from 2 months of age, and spatial memory in the water maze was evaluated 6 months after administration. Four small molecules, Example 23, Example 24, Example 25, and Example 26, were tested. The results are shown in Figures 40a-40b. Compared with the WT group, the model group had a significantly weakened spatial memory ability, and the improvement of spatial memory ability by Example 25 was the most obvious, based on indicators such as the number of round trips to the escape platform and the time spent in the target quadrant.

[0317] Experiment 17. AD mouse light / dark box Experimental and analytical methods: The transgenic mice used in this experiment were transgenic mice introduced with APP / P Example 13B, and the background mouse was C57BL / 6J. The phenotype of this model mouse was cognitive behavioral changes at 3 months of age, senile lentigo at 5 months of age, and formation of a large number of senile lentigo at 12 months of age. The principle of the light-dark box experiment is that mice have a natural tendency to like dark places, so after being placed in the light box, they will enter the dark box that is connected to them voluntarily. However, in this experiment, the dark box is equipped with an electric stimulation device, and when the mouse enters the dark box, it will be shocked. Then, normal mice will not enter the dark box after being shocked several times, but AD mice will continue to enter the dark box because they cannot remember being shocked when they enter the dark box. Therefore, the latency before the mouse voluntarily enters the dark box after being placed in the light box reflects the memory strength of the mouse regarding electric shock, and the longer it takes to enter the dark box, the stronger the memory of electric shock. Results: In this experiment, the mice were administered from 2 months of age, and the spatial memory of the water maze was evaluated 6 months after administration. Four small molecules, namely, Example 23, Example 24, Example 25 and Example 26, were tested. The results are shown in Figure 41. Compared with the WT group, the memory of the electric shock in the model group was obviously weakened, while the small molecules of Example 23 and Example 25 can obviously enhance the memory of the electric shock in the model mice.

[0318] Experiment 18. TDP43 A315T Mouse survival time Experimental and analytical methods: In this experiment, A315T Transgenic mice were used. Mice of this line can simulate the onset of ALS (amyotrophic lateral sclerosis). Mice of this line usually begin to die from 90 days, reaching a median death rate of around 120 days. In this experiment, the mice were divided into two groups: a model group and an administration group. The model group was not administered, while the administration group was administered starting from the 60th day, and a test of small molecule Example 13B was performed at a dose of 40mpk. There were 20 mice per group, and in this experiment, the date of death and weight of each mouse were recorded, and weight curves and survival curves were plotted. Results: Body weight is an important index for reflecting the progression of ALS. As shown in FIG. 42a, the body weight of the mice in the model group began to decrease significantly from the 13th week, while that of the mice in the treatment group began to decrease significantly from the 16th week. As shown in the survival curve in FIG. 42b, the mice in the model group started to die in large numbers from the 90th day, with the median death rate being around 120 days, and all of the mice died in about 140 days. Meanwhile, the mice in the treatment group started to die from the 100th day, with large numbers of deaths from the 120th day, with the median death rate being 135 days, and all of the mice died in 150 days. As a result, the small molecule Example 13B suppressed the progression of ALS by suppressing the progression of TDP43. A315T The survival time of the transgenic mice could be effectively extended, suggesting that the small molecule may be able to effectively extend the survival time of ALS patients.

[0319] Experiment 19. SOD G93A Mouse gait analysis Experimental and analytical methods: In this experiment, SOD G93A Transgenic mice were used. Mice of this line can simulate the onset of ALS (amyotrophic lateral sclerosis), and with age, they show obvious motor dysfunction. Gait analysis is a behavioral analysis method for testing motor dysfunction. Mice with motor dysfunction have a significantly reduced stride length. In this experiment, the mice were divided into two groups: a model group and an administration group. The model group was not administered, and the administration group was administered starting from the 60th day, and a test of small molecule Example 13B was performed, with a dose of 40mpk. There were 10 mice per group, and the stride length of each mouse was measured at the 19th week. Results: As shown in FIG. 43, the stride length of the treated mice was much longer than that of the model group, suggesting that the small molecule Example 13B significantly improved the motor ability of SODG93A mice.

[0320] Experiment 20. SOD G93A Mouse open field test Experimental and analytical methods: In this experiment, SOD G93ATransgenic mice were used. Mice of this line can simulate the onset of ALS (amyotrophic lateral sclerosis), and with age, they show obvious motor dysfunction. Calculating the average movement speed in an open field is a behavioral analysis method for measuring motor dysfunction. The average movement speed of mice with motor dysfunction is significantly reduced. In this experiment, the mice were divided into three groups: a WT group, a model group, and an administration group. The WT group was normal mice, and the model group was not administered, while the administration group was started from the 60th day, and a test of small molecule Example 13B was performed, with a dose of 40mpk. There were 10 mice per group, and the average movement speed of each mouse in the open field was measured at regular intervals every week from the 14th week to the 19th week. Results: As shown in FIG. 44, the average movement speed of the mice in the administration group was much faster than that of the mice in the model group during the 6-week period measured, indicating that the small molecule Example 13B is an effective SOD inhibitor. G93A It was suggested that the mice's motor skills were significantly improved.

[0321] Experiment 21. DSS mouse colitis model Experimental and analytical methods: The purpose of this experiment is to investigate the therapeutic effect of multiple oral intragastric administration of test agents (Example 13B, Example 33, Example 34) on 3.0% dextran sulfate sodium (DSS)-induced ulcerative colitis in mice. Sixty healthy mice were divided into six groups, an average of 10 mice per group, according to body weight: normal control group, model group, positive drug group, and different test agent (Example 13B, Example 33, Example 34, all 80mpk) group. Mice other than the normal control group were allowed to drink 3.0% DSS solution ad libitum, and modeling was performed by administering it continuously for 8 days. Mice in the normal control group were allowed to drink autoclave-filtered water ad libitum, and modeling was performed by administering it continuously for 8 days. Administration began on the day of modeling, and the test agent was orally administered intragastrically once a day for 9 consecutive days according to the designed dose. During the administration period, the mouse body weight was monitored every day, and the shape of the mouse feces and the presence or absence of bleeding were observed. The day after the final administration, after deep anesthesia with CO2, blood was drawn from the heart, plasma was collected, and frozen at -80°C. Next, the abdominal cavity was incised, the liver was removed, and frozen at -80°C. Finally, the entire colon tissue was removed, the length of the colon was measured, and photographed. After dissection of the colon along the mesenteric side, the colon contents were washed with saline and weighed. Some of the lesion tissues were fixed in 10% formalin, stained with HE, and histopathological observations were performed, and the other colon tissues were frozen at -80°C. Results: (1) As shown in Figure 45a, the effect on the general condition of DSS colitis mice: After 4 days of modeling with 3.0% DSS, the modeling animals showed abnormalities such as loose stools, bloody stools, and weight loss, which then became more severe. The model group mice showed symptoms such as loss of appetite, fatigue, lethargy, piloerection, and lack of fur. With 720 mg / kg positive drug SASP and the control (Example 13B, Example 33, Example 34), some improvement was observed, but at the end of the experiment, abnormal conditions such as weight loss, loose stools, and bloody stools still existed. (2) As shown in Figure 45b, the effect on the weight and length of the large intestine of DSS colitis mice: After the mice were allowed to drink 3.0% DSS solution ad libitum, the large intestine of the mice showed atrophy, and the length of the large intestine was shortened and the weight was reduced. Both the positive drug and the control could inhibit the atrophy of the mouse large intestine. All of the tested reagents (Example 13B, Example 33, and Example 34) were able to increase the weight and length of the large intestine in DSS mice and inhibit large intestinal atrophy in mice.

[0322] Experiment 22. TNBS mouse colitis model Experimental and analytical methods: The purpose of this experiment is to examine the therapeutic effect of multiple oral intragastric administration of the test agents (Example 13B, Example 36, Example 37, all 80mpk) on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ulcerative colitis in mice. Rats other than the normal control group were fasted for 24 hours (not deprived of water), anesthetized with isoflurane, and then 0.5mL / rat of TNBS ethanol solution (TNBS 18mg / rat) was injected into the rectum using a latex hose, with the insertion length into the rectum being approximately 8cm. After removing the hose, the rats were held in a tilted position for 15 minutes while maintaining the anesthetized state. The normal control group rats were not stimulated by inserting the hose into the rectum. When the animals woke up, they were randomly divided into groups, and drug administration was started immediately (recorded as D1), and the blank group and model group were orally intragastricly administered an equal amount of solvent once a day for 7 consecutive days (D1 to D7). The condition of the animals and fecal status (soft stool, bloody stool) were observed daily, and body weight was also monitored. On the day after the final administration (D8), after deep anesthesia with CO2 and sacrifice, the animals were dissected, the entire colon tissue was removed, the colon was dissected along the mesenteric side, the colon contents were washed with saline, the weight and colon length were measured, the ulcer surface was measured with Image J software, the colon status was observed, and photographs were taken. The lesion tissue was fixed in formalin, stained with HE, and histopathological observation was performed. Results: (1) As shown in Figure 46a, the effect on the general condition of rats with TNBS-induced ulcerative colitis: the rats had loose stools the day after modeling with TNBS. The modeling rats had symptoms such as loss of appetite, fatigue, lethargy, piloerection, and lack of fur. The feces of some rats improved in the later stage of drug administration, but some rats continued to have loose stools until the end of the experiment, and individual rats showed obvious abdominal distension in the later stage of the experiment. (2) As shown in Figure 46b, the effect on the weight and length of the large intestine of rats with TNBS-induced colitis: According to the overall results on the large intestine weight per unit length of rats (large intestine weight / large intestine length) and the area of ​​large intestine ulcers, all of the test agents (Example 13B, Example 36, Example 37) could reduce the large intestine weight and large intestine ulcer area per unit length of rats, and Example 13B showed a more significant effect.

[0323] Experiment 23. Hypoglycemic control in DB mice Experimental and analytical methods: The obesity gene receptor (OB-R), also known as the diabetes gene (db), and the leptin receptor (Lepr) are closely related to obesity, hypertension, diabetes, and lipid metabolism disorders. The course of the disease is greatly influenced by genetic background. External insulin cannot suppress the increase in blood glucose and hepatic gluconeogenesis levels. Using genome editing technology and embryo injection technology, Lepr gene mutant mice were constructed. According to the results of blood glucose monitoring of the homozygous strain, its blood glucose level was significantly different from the wild control group, and it can be used for type 2 diabetes research. The blood glucose level of this strain of mice rose sharply from 8 weeks of age, reaching 30 mmol / L. Meanwhile, the blood glucose level of normal background mice was only about 6 mmol / L. From 8 weeks of age, the small molecules of Example 28, Example 29, and Example 30 were intragastrically administered once a day at a dose of 80 mpk. Mice were grouped into 20 mice and their blood glucose levels were monitored weekly from 8 weeks of age until sacrifice at 20 weeks of age. Results: As shown in FIG. 47, both Example 28 and Example 29 showed some degree of hypoglycemic effect, but Example 30 had the best hypoglycemic effect, which could reach the level of normal background mice.

[0324] Experiment 24. Weight loss and body fat reduction in DIO mice Experimental and analytical methods: The DIO model is an obesity model caused by high-fat diet administration. In this experiment, two strains of mice, C57BL / 6J and C57BL / 6N, were used and a high-fat diet containing 60% fat was adopted. When the mice were 7 weeks old, they were modeled with a high-fat diet, and fat diet administration was continued for 5 months until the mice's body weight reached 50g or more. Drug administration was started, body weight was recorded every week, and body fat percentage was measured once every 2 weeks using an echo MRI small animal body composition analyzer. A test was conducted to reduce body weight of five types of small molecules, Example 33, Example 34, Example 35, Example 36 and Example 37, by orally administering them once every 2 days at a dose of 80mpk. Results: As shown in Figures 48a to 48d, Example 34 and Example 35 had the best diet effect, and the body weight of the obese model mouse was reduced from 50g to about 35g, the normal mouse level. Example 33, Example 36, and Example 37 also had diet effect, and the weight was reduced to about 40g. In terms of body fat percentage, all small molecules can effectively burn fat in the mouse body and reduce the body fat percentage, but Example 34 and Example 35 had the best fat burning effect, followed by Example 33, Example 36, and Example 37.

[0325] Experiment 25. Raji-Luc hematoma cell inoculation into NOD-Scid immunodeficient mice Experimental and analytical methods: The purpose of this experiment is to test the in vivo killing effect of six kinds of small molecules on hematomas. In this experiment, 8-week-old male NOD-Scid immunodeficient mice were used as the inoculated mice of Raji-Luc cells, and a total of 80 mice were used. On the 9th day after inoculation, live fluorescence imaging was performed, and based on the fluorescence intensity, 60 mice with the most concentrated fluorescence intensity value in the middle region were selected for subsequent experiments. After the first live imaging, administration began, and the mice were intragastrically administered once a day at a dose of 100mpk. The test small molecules were Example 25A, Example 25B, Example 28, Example 38, Example 39, and Example 40, with 10 mice per group. After the start of administration, live imaging was performed twice a week to observe the progression of tumors in the whole blood of the mice. Results: As shown in Figure 49, the fluorescence intensity of the control group was very high, while the fluorescence intensity of the treatment group was obviously reduced. Among them, the treatment groups of Example 28 and Example 38 were too toxic, so all of them died on the 21st day, and the fluorescence value could not be obtained. In addition, Example 25A, Example 25B, Example 39, and Example 40 all showed good tumor killing effect.

[0326] Experiment 26. Cell proliferation measurement using small molecule compounds Experimental and analytical methods: Cells were seeded into a capped 96-well plate, and the highest concentration of the test compound was 10 μM, and then diluted 3-fold into a series of test concentrations, and the cells were incubated at 37°C, 5% CO2 for 72 hours. The DMSO concentration was 0.5%. 100 ul of medium was taken out from each well. 100 ul of reagent (Celltitute Glo Analysis Kit) was added to each well, and the plate (protected from light) was shaken for 2 minutes on a plate shaker. The plate (protected from light) was incubated for 30 minutes at room temperature. The chemiluminescence values ​​were recorded by a multiplate reader, and the experimental results are shown below:

[0327] [Table 13]

[0328] In the table, analysis was performed in cell proliferation experiments using 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 the inhibitory effect of the compounds of the present invention on lymphoma cell proliferation. Among them, Example 2A had the best inhibitory activity against U937, and Example 13A had the best inhibitory effect on the proliferation of OCI-LY3 cell line and HT cells. "-" in the table indicates that no data is available.

[0329] Experiment 27. PD mouse model Experimental and analytical methods: In this experiment, mice of C57BL / 6 strain were used and divided into five groups: a normal control group (without MPTP injection), a model group (modeling with MPTP), and a treatment group with three types of small molecule compounds (Example 13B, Example 25A, Example 25B). Administration was started at the same time as modeling. Modeling continued for 5 days, intraperitoneally injected at 30mpk, and administration continued for 15 days at 80mpk. At the end point, the brain was removed and collected after cardiac perfusion, and immersed in PFA for fixation. After fixation was completed, it was transferred to sucrose for dehydration, and after complete dehydration, it was embedded in OCT and sliced ​​for TH staining. The measurement index was the number of TH-positive cells in the substantia nigra. The more positive cells there were, the better the protective effect of the small molecule against MPTP injury. Results: 12 mice per group, statistical results are shown in Figure 50, T-test results are **: p<0.01, #: p<0.05. Compared with the model group, only the Example 13B group among the three small molecules has statistical difference, which shows that Example 13B has good protective effect on the substantia nigra neurons.

[0330] Experiment 28. Pseudotyped SARS-CoV-2-infected cell model Experimental and analytical methods: First, a pseudotyped new coronavirus with infectious but non-replicative capabilities was cultured using HEK293T cells. After collecting the pseudotyped virus from the supernatant, normal HEK293T cells with ACE2 receptors on their surface were used as the infection target and infected with the pseudotyped virus. After infection with the virus, the mitochondria of HEK293T cells were fragmented, which could be monitored by mitochondrial fluorescent staining (mitotracker red), indicating that the cells were destroyed by the pseudotyped virus, and this was used as the model group. Meanwhile, in the experimental group, 300 nM of Example 13B was used to incubate the cells with the pseudotyped virus for 1 hour in advance, and the protective effect of the small molecule Example 13B against viral infection was confirmed. Results: As shown in Figure 51, compared with the normal control group (without pseudotyped virus infection), the mitochondria in the model group cells were obviously fragmented, while the mitochondria in the experimental group cells were not fragmented, suggesting that the small molecule Example 13B has an excellent protective effect against pseudotyped new coronavirus infection.

[0331] Experiment 29. Rat TBI acute traumatic brain injury model Experimental and analytical methods: In this experiment, a traumatic brain injury (TBI) rat model was established using male SD rats, and randomly divided into a solvent control group (8 rats), experimental example 13B (80 mpk, 10 rats), and experimental example 26 (80 mpk, 9 rats). Administration began after TBI surgery, and was administered intragastrically once a day for 7 consecutive days. At the end of the experiment (day 7), brain tissue was collected to measure the percentage of cell apoptosis in the hippocampus and adjacent areas of injury. Results: The flow cytometry analysis shown in Figure 52 showed that the initial apoptosis rate and total apoptosis rate of rats in the experimental group of Example 13B (80mpk) and Example 26 (80mpk) were significantly lower than that of the solvent control group (P<0.05). This experimental system showed that multiple intravenous administration of Example 13B and Example 26 can significantly improve the neurological function damage and brain cell apoptosis of acute traumatic brain injury (TBI) rats, and promote the recovery of motor ability and neurological function.

[0332] Experiment 30. In vitro killing effect on pancreatic adenocarcinoma cells Experimental and analytical methods: Mia Paca-2 cells were used in this study. This cell line is a pancreatic adenocarcinoma cell line and has been widely used in drug sensitivity experiments. Drug sensitivity experiments were performed on 11 small molecules, with the highest concentration of 10uM and diluted 3-fold to obtain a 7-point concentration gradient. Celltiter glo was used to measure cell activity, and dose-response curves were plotted using Graphpad Prism. The obtained drug sensitivity curves are shown in the figure below. Results: As shown in the drug sensitivity curve in Figure 53, the lower the IC50 value, the higher the sensitivity to the small molecule. In other words, Mia Paca-2 cells were shown to be the most sensitive to Example 26A.

[0333] Experiment 31. Suppression of excessive urination in DB animals Experimental and analytical methods: The leptin receptor gene (OB-R) is also known as the diabetes gene (db), and the leptin receptor (Lepr) is closely related to obesity, hypertension, diabetes, and lipid metabolism disorders. The course of the disease is greatly influenced by genetic background. External insulin cannot suppress the increase in blood glucose and hepatic gluconeogenesis levels. Using genome editing technology and embryo injection technology, Lepr gene mutant mice were constructed. According to the results of blood glucose monitoring of the homozygous strain, its blood glucose level was significantly different from the wild control group, and it can be used for type 2 diabetes research. This strain of mouse has a large amount of urine, about 100 times that of normal mice, so it can simulate patients with frequent urination. In this experiment, the mice were divided into three groups: a model group (non-administered, mice with high urine volume), a control group (non-administered, normal mice without disease), and an experimental group (intragastric administration of Example 1A, 80mpk once every two days), with two mice in the control group and three mice in the other groups. Example 1A was used as the small molecule. After administering the drug continuously for 5 weeks from the 8th week, the urine volume of mice individually housed in cages was observed in the cage. The larger the area wetted by urine in the cage, the larger the urine volume of the mouse in the cage. Results: As shown in the cage picture in Figure 54, the area of ​​urine wetted in the cage of the three model mice was the largest, the area of ​​urine wetted in the cage of the control mice was almost not observed, and the area of ​​urine wetted in the cage of the drug administration group mice was obviously reduced compared to the model group mice. This shows that Example 1A has the function of suppressing frequent urination.

[0334] Experiment 32. Attenuating effect of high-fat diet on cardiovascular inflammation in APOE mutant mice. Experimental and analytical methods: After purchasing 6-week-old male APOE- / - mutant B6J mice and WT B6J mice, they were adapted for 2 weeks in a breeding room. When the mice were 8 weeks old, the groups except for the vehicle group and the WT group were administered a high-fat diet (Research Diets, D12492). When the mice were 9 weeks old, the control group was administered a 10% cyclodextrin solution, and the other groups were started to be orally intragastrically administered different small molecule compounds (Example 13B, Example 33, Example 36 and Example 37) once every 2 days. After 16 weeks of administration of the high-fat diet, the APOE mice were sacrificed and the aortic arches were dissected and collected. A constant mass of 100 mg / animal aortic vascular epithelial tissue was subjected to MSD multifactor measurement to confirm the expression levels of various inflammatory factors including IL10, IL-1β and KC / GRO. Results: As shown in Figures 55a to 55c, the three inflammatory factors tended to decrease under the intervention of small molecules, with the most obvious decrease observed in Example 13B, indicating that the small molecules have anti-cardiovascular inflammatory effects.

[0335] Experiment 33. Pharmacodynamic experiment of Example 13B on subthreshold hypnotic doses of sodium pentobarbital affecting sleep in mice Experimental and analytical methods: To determine whether the subject had a hypnotic effect, the effects of a single oral intragastric administration of Example 13B on the time to sleep onset (sleep latency) and duration of sleep induced by a subthreshold hypnotic dose of sodium pentobarbital in ICR mice were measured, and a pharmacodynamic comparison was made with diazepam, a positive control drug. After the mice were reared for adaptation, 50 animals that met the criteria were selected based on their weight and included in the experimental group, and divided into five groups of 10 animals per group using Excel's complete random grouping method: blank control group, positive control group (diazepam), high, medium, and low dose groups. One hour after administering the appropriate drug solution and vehicle to the animals in each group, the animals in each group were intraperitoneally injected with the maximum subthreshold dose of sodium pentobarbital (a preliminary experiment was conducted in advance to determine the maximum subthreshold hypnotic dose of sodium pentobarbital), and the time it took each animal to fall asleep (sleep latency), the number of animals that fell asleep within 30 minutes (animals that lost the righting reflex for 1 minute or more), and the duration of sleep for each mouse were recorded. Results: As shown in Figures 56a and 56b, diazepam 1 mg / kg could obviously shorten the sleep latency of mice. Compared with the blank control group, all the small molecules shown in the figures could shorten the sleep latency of mice in a dose-dependent manner, but no statistically significant difference was observed. In addition, the effect of the small molecules shown in the figures on the sleep latency of mice was obviously weaker than that of diazepam 1 mg / kg. Compared with the blank control group, when diazepam 1 mg / kg was administered to the positive control group, the sleep of mice could obviously be extended. Compared with the blank control group, the small molecules shown in the figures could extend the sleep time of mice, but no statistically significant difference was observed. In addition, the effect of the small molecules shown in the figures on extending the sleep time of mice was obviously weaker than that of diazepam 1 mg / kg. [Industrial Applicability]

[0336] Although the present invention has been described herein with reference to specific embodiments, it will be apparent that various modifications and alterations may still be made without departing from the spirit and scope of the present invention. Accordingly, the present specification is to be understood as illustrative and not restrictive.

Claims

1. The structural formula of the derivative is shown in general formula (I): 【Chemistry 1】 The derivative represented by the general formula (I) is a compound in which the following fragment A and fragment B are bonded together: 【Chemistry 2】 where Z is NR 1 R 2 and R 1 and R 2 each independently represents hydrogen, substituted or unsubstituted C 1 -C 10 is an alkyl group, C 1 -C 10 The substituents of the alkyl group are halogen, hydroxyl group, amino group, nitro group, cyano group, C 1 -C 4 Alkyl group, C 1 -C 4 Halogen-substituted alkyl groups, C 1 -C 4 Alkoxy group, C 3 -C 6 Cycloalkyl groups, C 2 -C 4 Alkenylene group, C 2 -C 4 Alkynyl group, phenyl group, benzyl group, C 3 -C 14 Heterocyclic groups, C 3 -C 14 heteroaryl groups, the heteroatoms being selected from one or more of N, O, and S, or R 1 and R 2 and form a 3- to 8-membered ring, and the 3- to 8-membered ring is 1 -C 10 Alkyl group, C 3 -C 10 Cycloalkyl groups, C 6 -C 20 Aryl group, C 3 -C 14 Heteroaryl groups, halogens, hydroxy groups, amino groups, alkoxy groups, -CF 3 ,-SCIENCE FICTION 5 or the heteroatom is sulfur, oxygen, NH or NR a wherein the heteroatom is one or more of N, O, and S; X is C(O) or S(O) 2 and Y is C(R d )(R e ), C(O) or S(O) 2 and R d , R e are independently hydrogen or C having at least one substituent 1 -C 10 Alkyl group, C 3 -C 10 Cycloalkyl groups, C 6 -C 20 Aryl group, or C 3 -C 14 Heteroaryl groups, the substituents of which are halogen, hydroxy, amino, nitro, cyano, aldehyde, carboxyl, alkoxy, -CF 3 or -SF 5 and the heteroatom is selected from one or more of N, O, and S, or R d and R e and form a 3- to 8-membered ring, and the 3- to 8-membered ring is 1 -C 10 Alkyl group, C 3 -C 10 Cycloalkyl groups, C 6 -C 20 Aryl group, C 3 -C 14 Heteroaryl groups, halogens, hydroxy groups, amino groups, alkoxy groups, -CF 3 ,-SCIENCE FICTION 5 or the heteroatom is sulfur, oxygen, NH or NR a wherein the heteroatom is one or more of N, O, and S; X 2 is O, S or NH, R a are independently hydrogen or C having at least one substituent 1 -C 10 Alkyl group, C 3 -C 10 Cycloalkyl groups, C 6 -C 20 Aryl group, or C 3 -C 14 Heteroaryl groups, the substituents of which are halogen, hydroxy, amino, nitro, cyano, alkoxy, -CF 3 or -SF 5 and the heteroatom is selected from one or more of N, O, and S; n is an integer from 0 to 10, and n is not 0, and 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, and m is 0; R 3 , R 4a , R 4b , R 5a , R 5b are each independently hydrogen or selected from a halogen, a substituted alkyl group, a hydroxyl group, and an amino group; 【Chemistry 3】 represents a single or double bond, each 【Chemistry 4】 The use of derivatives based on the sarsasapogenin structure, characterized by independently exhibiting racemic, S or R configuration, for preparing a therapeutic agent for related diseases resulting from mitochondrial dysfunction.

2. The use according to claim 1, characterized in that the structural formula of the derivative is shown in general formula (II): 【Chemistry 5】

3. The use according to claim 1, characterized in that the structural formula of the derivative is as shown in general formula III. 【Chemistry 6】

4. The use according to claim 1, characterized in that the structural formula of the derivative is shown in general formula IV. 【Chemistry 7】

5. The structural formula of the derivative is shown in general formula V: 【Chemistry 8】 Here, R 6 is hydrogen, substituted or unsubstituted C 1 -C 10 is an alkyl group, C 1 -C 10 The substituents of the alkyl group are halogen, hydroxyl, -NH 2 , nitro group, -CN, C 1 -C 4 Alkyl group, C 1 -C 4 Halogen-substituted alkyl groups, C 1 -C 4 Alkoxy group, C 3 -C 6 Cycloalkyl groups, C 2 -C 4 Alkenylene group, C 2 -C 4 Alkynyl group, phenyl group, benzyl group, pyridinyl group, -CO alkyl group, -CO aryl group, -SO 2 Alkyl group, -SO 2 Aryl group, -CO 2 Alkyl group, C 2 -C 4 (CO) alkenyl group, -CO 2 Aryl group, -SO 3 Selected from H, L is hydrogen, substituted or unsubstituted C 1 -C 10 is an alkyl group, C 1 -C 10 The substituents of the alkyl group are halogen, hydroxyl, -NH 2 , nitro group, -CN, C 1 -C 4 Alkyl group, C 1 -C 4 Halogen-substituted alkyl groups, C 1 -C 4 Alkoxy group, C 3 -C 6 Cycloalkyl groups, C 2 -C 4 Alkenylene group, C 2 -C 4 alkynyl groups, n is an integer from 0 to 10, n2 is 0, 1, 2, or 3; m,m' are independently 1, 2, 3, or 4; W 1 is C or NH, V 1 is C or NH, 2. The use according to claim 1, characterized in that M is C, S, O or NH.

6. The structural formula of the derivative is shown in general formula VI: 【Chemistry 9】 Y 1 is C(Rd)(Re), C(O) or S(O) 2 Rd and Re are independently hydrogen or C having at least one substituent. 1 -C 10 Alkyl group, C 3 -C 10 Cycloalkyl groups, C 6 -C 20 Aryl group, or C 3 -C 14 Heteroaryl groups, the substituents of which are halogen, hydroxy, amino, nitro, cyano, aldehyde, carboxyl, alkoxy, -CF 3 Or-SF 5 and the heteroatom is selected from one or more of N, O, and S, or R d and R e and form a 3- to 8-membered ring, and the 3- to 8-membered ring is 1 -C 10 Alkyl group, C 3 -C 10 Cycloalkyl groups, C 6 -C 20 Aryl group, C 3 -C 14 Heteroaryl groups, halogens, hydroxy groups, amino groups, alkoxy groups, -CF 3 ,-SCIENCE FICTION 5 or the heteroatom is sulfur, oxygen, NH or NR a wherein the heteroatom is one or more of N, O, and S; L is hydrogen, substituted or unsubstituted C 1 -C 10 is an alkyl group, C 1 -C 10 The substituents of the alkyl group are halogen, hydroxyl, -NH 2 , nitro group, -CN, C 1 -C 4 Alkyl group, C 1 -C 4 Halogen-substituted alkyl groups, C 1 -C 4 Alkoxy group, C 3 -C 6 Cycloalkyl groups, C 2 -C 4 Alkenylene group, C 2 -C 4 alkynyl groups, n is an integer from 0 to 10, n2 is 0, 1, 2, or 3; n3 is an integer from 1 to 10, 2. Use according to claim 1, characterized in that m is an integer from 0 to 10.

7. The structural formula of the derivative is shown in general formula VII: 【Chemistry 10】 Here, R 6 , R 7 are independently hydrogen, substituted or unsubstituted C 1 -C 10 is an alkyl group, C 1 -C 10 The substituents of the alkyl group are halogen, hydroxyl, -NH 2 , nitro group, -CN, C 1 -C 4 Alkyl group, C 1 -C 4 Halogen-substituted alkyl groups, C 1 -C 4 Alkoxy group, C 3 -C 6 Cycloalkyl groups, C 2 -C 4 Alkenylene group, C 2 -C 4 Alkynyl group, phenyl group, benzyl group, pyridinyl group, -CO alkyl group, -CO aryl group, -SO 2 Alkyl group, -SO 2 Aryl group, -CO 2 Alkyl group, C 2 -C 4 (CO) alkenyl group, -CO 2 Aryl group, -SO 3 Selected from H, L is hydrogen, substituted or unsubstituted C 1 -C 10 is an alkyl group, C 1 -C 10 The substituents of the alkyl group are halogen, hydroxyl, -NH 2 , nitro group, -CN, C 1 -C 4 Alkyl group, C 1 -C 4 Halogen-substituted alkyl groups, C 1 -C 4 Alkoxy group, C 3 -C 6 Cycloalkyl groups, C 2 -C 4 Alkenylene group, C 2 -C 4 alkynyl groups, W 2 is C or NH, V 2 is C, O, S or NH, n is an integer from 0 to 10, n1 is an integer from 1 to 10, 2. The use according to claim 1, characterized in that n2 is 0, 1, 2 or 3.

8. The structural formula of the derivative is shown in general formula VIII: 【Chemistry 11】 Z 1 is hydrogen, substituted or unsubstituted C 1 -C 10 is an alkyl group, C 1 -C 10 The substituents of the alkyl group are halogen, hydroxyl, -NH 2 , nitro group, -CN, C 1 -C 4 Alkyl group, C 1 -C 4 Halogen-substituted alkyl groups, C 1 -C 4 Alkoxy group, C 3 -C 6 Cycloalkyl groups, C 2 -C 4 Alkenylene group, C 2 -C 4 Alkynyl group, phenyl group, benzyl group, pyridinyl group, -CO alkyl group, -CO aryl group, -SO 2 Alkyl group, -SO 2 Aryl group, -CO 2 Alkyl group, C 2 -C 4 (CO) alkenyl group, -CO 2 Aryl group, -SO 3 Selected from H, W 3 is C, O, S or NH, n is an integer from 0 to 10, 2. The use according to claim 1, characterized in that n4, n5, n6, n7 are independently integers from 1 to 4.

9. 2. The use according to claim 1, characterized in that fragment B in the structural formula of the derivative has the following structure: 【Chemistry 12】

10. 2. The use according to claim 1, characterized in that the derivative is either a compound represented by the formula: 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemical 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemical 27】 【Chemistry 28】 【Chemical 29】 【Chemistry 30】 【Chemistry 31】 【Chemistry 32】 【Chemical 33】 【Chemical 34】 【Chemistry 35】 【Chemical 36】 【Chemical 37】 【Chemical Formula 38】 【Chemical 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 【Chemistry 46】 【Chemistry 47】 【Chemistry 48】

11. 2. The use according to claim 1, characterized in that one or more hydrogen atoms in the compound are deuterium atoms.

12. The pharmaceutical composition comprises a first component and a pharma- ceutically acceptable carrier, or a combination of a first component, a first component and a pharma- ceutically acceptable carrier, wherein the first component is a compound, a pharma- ceutically acceptable salt, a stereoisomer, or a tautomer thereof, according to any one of claims 1 to 11, Use of a pharmaceutical composition for preparing a medicament for treating a disease associated with mitochondrial dysfunction, wherein the second component is an additional therapeutic agent comprising an antidepressant, an antimanic drug, a Parkinson's drug, an Alzheimer's drug, or a combination thereof.

13. 13. Use according to claim 12, characterized in that the pharma- ceutically acceptable salt is selected from one or more of the following salts: hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, triflate, benzenesulfonate, p-toluenesulfonate (toluenesulfonate), 1-naphthalenesulfonate, 2-naphthalenesulfonate, acetate, trifluoroacetate, malate, tartrate, citrate, lactate, oxalate, succinate, fumarate, maleate, benzoate, salicylate, phenylacetate, mandelate. Preferably, the additional therapeutic agent is moclobemide, toloxatone, fluoxetine, paroxetine, citalopram, sertraline, venlafaxine, trimipramine, trazodone, imipramine, desipramine, clomipramine, amitriptyline, nortriptyline, doxepin, maprotiline, loxapine, amoxapine, mirtazapine, buspirone, chlormezanone, tandospirone, lithium carbonate, tacrine, huperzine A, galantamine, donepezil, rivastigmine, memantine, pramipexole, talipexole, ropinirole, or a combination thereof.

14. The use according to any one of claims 1 to 13, characterized in that the associated diseases caused by mitochondrial dysfunction include metabolic diseases, tumors, inflammation and central nervous system diseases.

15. The metabolic diseases include hyperglycemia, hyperlipidemia, hypercholesterolemia, high low-density lipoprotein, low high-density lipoprotein, angiogenesis diseases, non-alcoholic fatty liver disease, cerebrovascular disease, myocardial infarction, atherosclerosis, coronary artery disease, anti-aging, urinary urgency / frequent urination, type I diabetes, and chronic obstructive pulmonary disease; The tumors include benign prostatic hyperplasia, Wegener's granulomatosis, pulmonary sarcoidosis, leukemia, lymphoma, pancreatic adenocarcinoma, and neuronal tumors; The inflammation includes peripheral neuritis, chemotherapy-induced peripheral neuritis, autoimmune diseases, diseases associated with organ transplantation, influenza virus, coronavirus (prevention of infectious diseases, treatment, and removal of sequelae), 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, sicca syndrome, systemic sclerosis, antiphospholipid antibody syndrome, vasculitis, osteoarthritis, autoimmune hepatitis, autoimmune liver and gallbladder disease, primary sclerosing cholangitis, nephritis, gluten-sensitive enteropathy, autoimmune thrombocytopenia, transplant rejection, ischemia-reperfusion injury of solid organs, sepsis, periodontitis, systemic inflammatory response syndrome, myocarditis, allergic diseases, asthma, interleukin-I converting enzyme-associated fever syndrome, and Behcet's disease, The use according to claim 14, wherein the central nervous system disease comprises Pick's disease, spinal cord injury repair, depression, anxiety, Parkinson's disease, Alzheimer's disease, sleep disorder, sleep disorder, ischemic stroke, hemorrhagic stroke, amyotrophic lateral sclerosis, traumatic brain injury, brain atrophy, Huntington's disease, schizophrenia, manic state, drug withdrawal, multiple sclerosis, sleep improvement, and muscle weakness.

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