WO2023155757A1 - 基于菝契皂苷元结构的衍生物及其药物组合物的用途 - Google Patents

基于菝契皂苷元结构的衍生物及其药物组合物的用途 Download PDF

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WO2023155757A1
WO2023155757A1 PCT/CN2023/075727 CN2023075727W WO2023155757A1 WO 2023155757 A1 WO2023155757 A1 WO 2023155757A1 CN 2023075727 W CN2023075727 W CN 2023075727W WO 2023155757 A1 WO2023155757 A1 WO 2023155757A1
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alkyl
mice
group
disease
hydroxyl
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PCT/CN2023/075727
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English (en)
French (fr)
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王娜
翁建阳
南志远
孔凡胜
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北京清博汇能医药科技有限公司
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Priority claimed from CN202310093727.8A external-priority patent/CN116621912A/zh
Application filed by 北京清博汇能医药科技有限公司 filed Critical 北京清博汇能医药科技有限公司
Priority to AU2023221806A priority Critical patent/AU2023221806A1/en
Publication of WO2023155757A1 publication Critical patent/WO2023155757A1/zh

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/56Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/14Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16Anti-Parkinson drugs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/22Anxiolytics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/24Antidepressants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/08Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents

Definitions

  • the present invention relates to the field of medical technology, in particular to the field of chemical synthesis of medicines, and specifically refers to a novel method for preparing drugs based on smilagenin-like structure derivatives and pharmaceutical compositions thereof for the treatment of associated diseases caused by abnormal mitochondrial function. use.
  • Anemarrhena is one of the common Chinese medicines in my country. It is the dry tuber of Anemarrhena asphodeloides Bunge, a plant of the Liliaceae family. Its extract has been shown to have biological activities such as diuretic, antidiabetic, antiplatelet aggregation, antifungal, regulation of metabolism, and also exhibits inhibitory effect on cyclic AMP phosphodiesterase.
  • the main chemical components of the extract are steroidal saponins, diphenylpyrone, polysaccharides and lignin.
  • steroidal saponins include timosaponins A-I, A-II, A-III, A-IV, B-I, B-II and B-III, and malcogenin 3-O- ⁇ -D-glucopyranose Base (1 ⁇ 2)- ⁇ -D-galactopyranoside B, degalactoside, F-rissasapenoside and smilaxaponin, etc.
  • it also contains Anemarrhena polysaccharide A/B/C/D, cis-cypress resinol, monomethyl-cis-cypress resinol, oxidation-cis-cypress resinol, 2,6,4'-trihydroxy-4 -Methoxybenzophenone, p-hydroxyphenyl crotonoleic acid, vinyl behenicate, ⁇ -sitosterol, mangiferin, niacin, niacinamide and pantothenic acid, etc.
  • Mitochondrial function and behavior are central to human physiology. Mitochondria perform diverse and interrelated functions, producing ATP and many biosyntheses while also contributing to cellular stress responses such as autophagy and apoptosis. Mitochondria form a dynamic, interconnected network and are tightly bound together with other cellular compartments. Furthermore, mitochondrial function extends beyond cell boundaries and affects the physiology and organization of organisms by regulating intercellular communication.
  • the mitochondrial respiratory chain is mainly composed of mitochondrial respiratory chain enzymes, and the defect of the mitochondrial respiratory chain enzyme complex is an important cause of mitochondrial diseases (about 30%-40% of mitochondrial diseases are caused by the defects of mitochondrial respiratory chain enzymes).
  • the structure and function of the human respiratory chain super complex, oxidative phosphorylation is completed step by step by five respiratory chain protein complexes located on the inner mitochondrial membrane, and these five protein complexes are complex I (NADH dehydrogenase ), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (ATP synthase).
  • complex I NADH dehydrogenase
  • complex II succinate dehydrogenase
  • complex III cytochrome c reductase
  • complex IV cytochrome c oxidase
  • complex V ATP synthase
  • the action of small molecules on the mitochondria can maintain the complex in an active state, and allosterically regulate the activity and electron transfer efficiency of the complex, and at the same time contribute to the overall structure stability of the complex, thereby reducing the mitochondrial ROS (superoxide free base) generated.
  • mitochondrial ROS superoxide free base
  • Stroke is commonly known as stroke, including ischemic stroke (cerebral infarction) and hemorrhagic stroke (cerebral parenchymal hemorrhage, ventricular outflow blood, subarachnoid hemorrhage).
  • ischemic stroke Cerebral infarction
  • hemorrhagic stroke Cerebral parenchymal hemorrhage, ventricular outflow blood, subarachnoid hemorrhage
  • multiple causes of stroke lead to cerebrovascular damage, focal (or overall) brain tissue damage, causing clinical symptoms for more than 24 hours or death. It has the characteristics of high morbidity, disability, recurrence and mortality. Stroke is the leading cause of death among Chinese residents.
  • Amyotrophic lateral sclerosis also known as motor neuron disease, is a chronic, progressive degenerative disease of upper and lower motor neurons and the trunk, limbs, and head and face muscles they innervate. It often manifests as progressively aggravated muscle weakness, muscle atrophy, and muscle fasciculation caused by combined damage to upper and lower motor neurons.
  • the clinical manifestations are progressive skeletal muscle weakness, muscle atrophy, muscle fasciculation, bulbar palsy, and pyramidal tract signs. Early symptoms are mild and easily confused with other diseases. Patients may just feel some weakness, twitching, and easy fatigue. Some symptoms gradually progress to general muscle atrophy and dysphagia, and finally produce respiratory failure.
  • Neurodegenerative diseases are divided into acute neurodegenerative diseases and chronic neurodegenerative diseases.
  • Acute neurodegenerative diseases mainly include stroke, brain injury (BI) and epilepsy; chronic neurodegenerative diseases mainly include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), muscular atrophy Lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA) and Pick's disease, etc.
  • the causes of neurodegenerative diseases mainly include the following four aspects: 1. Oxidative stress. Oxidative stress is caused by the excessive production of free radicals and (or) the failure to remove them in time, the imbalance of oxidation and anti-oxidation in the body, and the damage of cells and tissues in the body.
  • Free radicals are atoms or groups with unpaired electrons, including hydroxyl radicals, superoxide anions, nitric oxide, etc.
  • oxidative damage to nerve tissue has been found in neurodegenerative diseases such as AD, PD, and ALS; 2.
  • Mitochondrial dysfunction There are mtDNA defects and abnormal oxidative phosphorylation in the brain of AD patients.
  • Polymerase chain reaction (PCR) and Western blot hybridization detected mtDNA breaks, base deletions and mistranslation mutations in the brain tissue of sporadic AD patients. Electron microscope observation confirmed that the number of mitochondria increased, the structure was abnormal, and lamellar bodies and crystalline inclusion bodies appeared.
  • Novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19), referred to as "new coronary pneumonia", named “2019 coronavirus disease” by the World Health Organization, refers to the acute respiratory infectious disease caused by 2019 new coronavirus infection.
  • the clinical manifestations of patients with pneumonia infected by the new coronavirus are: fever, fatigue, and dry cough as the main manifestations, and a state of hypoxia and hypoxia will occur. About half of the patients will have dyspnea after more than a week, and severe cases will rapidly progress to acute respiratory distress syndrome. , septic shock, refractory metabolic acidosis and coagulopathy.
  • the treatment drugs include Paxlovid, Azvudine Tablets, Monogravir Capsules, Sanhan Huashi Granules, Qingfei Paidu Decoction, etc.
  • the purpose of the present invention is to overcome the defects in the above-mentioned prior art.
  • the first aspect of the present invention provides a derivative based on the structure of smilagenin in the preparation of the treatment of related diseases caused by abnormal mitochondrial function.
  • the purposes in medicine, its main characteristic is, the structural formula of described derivative is as shown in general formula I,
  • the derivative represented by the general formula I is formed by linking the following fragment A and fragment B,
  • Z is NR 1 R 2 ;
  • R 1 and R 2 are each independently hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, and the substituents of C 1 -C 10 alkyl are selected from halogen, hydroxyl, amino , nitro, cyano, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 3 -C 6 cycloalkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, phenyl, benzyl, C 3 -C 14 heterocyclyl, C 3 -C 14 heteroaryl, one or more heteroatoms selected from N, O, S; or R 1 and R 2 together form a three- to eight-membered ring, and the three- to eight-membered ring has one or more selected from C 1 -C 10 alkyl, C 3 -C 10 cycloalkyl, C 6
  • X is C(O) or S(O) 2 ;
  • Y is C(R d )(R e ), C(O) or S(O) 2
  • R d and R e are each independently hydrogen or C 1 -C 10 alkyl having at least one substituent, C 3 -C 10 cycloalkyl, C 6 -C 20 aryl, or C 3 -C 14 heteroaryl, wherein the substituents are selected from halogen, hydroxyl, amino, nitro, cyano, aldehyde, carboxyl, alkoxy group, -CF 3 or -SF 5 , one or more heteroatoms selected from N, O, S, or, R d and Re form together a three to eight-membered ring, and the three to eight-membered ring has one or more selected from C1-C10 alkyl, C3-C10 cycloalkyl, C 6- C 20 aryl, or C 3 -C 14 heteroaryl, halogen, hydroxyl, amino, alkoxy, -CF 3 ,
  • X2 is O, S or NH
  • R a is independently hydrogen or C 1 -C 10 alkyl, C 3 -C 10 cycloalkyl, C 6 -C 20 aryl, or C 3 - having at least one substituent C 14 Heteroaryl, wherein the substituent is selected from halogen, hydroxyl, amino, nitro, cyano, alkoxy, -CF 3 or -SF 5, and the heteroatom is selected from one or more of N, O, S ;
  • n is an integer from 0 to 10 and n is not 0, m is 1, or n is 0 and m is 1, or n is an integer from 0 to 10 and n is not 0, m is 0;
  • R 3 , R 4a , R 4b , R 5a , R 5b are each independently hydrogen or selected from halogen, substituted alkyl, hydroxyl, amino;
  • Each "*" independently represents the racemic, S or R configuration.
  • R 6 is hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, and the substituent of C 1 -C 10 alkyl is selected from halogen, hydroxyl, -NH 2 , nitro, -CN, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 3 -C 6 cycloalkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, phenyl, Benzyl, pyridyl, -COalkyl, -COaryl, -SO2alkyl , -SO2aryl , -CO2alkyl, C2 - C4 (CO)alkenyl, -CO2aryl , -SO 3 H;
  • L is hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, the substituent of C 1 -C 10 alkyl is selected from halogen, hydroxyl, -NH 2 , nitro, -CN, C 1 -C 4 alkyl , C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 3 -C 6 cycloalkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl;
  • n is an integer from 0 to 10;
  • n2 is 0, 1, 2, or 3;
  • n, m' are independently an integer from 1 to 4.
  • W 1 is C or NH
  • V 1 is C or NH
  • M is C, S, O or NH
  • Y 1 is C(Rd)(Re), C(O) or S(O) 2
  • Rd, Re are independently hydrogen or C 1 -C 10 alkyl with at least one substituent, C 3 -C 10 ring Alkyl, C 6 -C 20 aryl, or C 3 -C 14 heteroaryl, wherein the substituent is selected from halogen, hydroxyl, amino, nitro, cyano, aldehyde, carboxyl, alkoxy, -CF 3 or -SF 5 , one or more heteroatoms selected from N, O, and S, or, R d and Re form together a three- to eight-membered ring, and the three- to eight-membered ring has one or more from C 1 -C 10 alkyl, C 3 -C 10 cycloalkyl, C 6 -C 20 aryl, or C 3 -C 14 heteroaryl, halogen, hydroxyl, amino, alkoxy, -CF 3 , -SF 5 or
  • L is hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, the substituent of C 1 -C 10 alkyl is selected from halogen, hydroxyl, -NH 2 , nitro, -CN, C 1 -C 4 alkyl , C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 3 -C 6 cycloalkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl;
  • n is an integer from 0 to 10;
  • n2 is 0, 1, 2, or 3;
  • n3 is an integer from 1 to 10
  • n is an integer of 0 to 10.
  • R 6 and R 7 are independently hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, C 1 -C 10 alkyl substituents are selected from halogen, hydroxyl, -NH 2 , nitro, -CN , C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 3 -C 6 cycloalkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkyne Base, phenyl, benzyl, pyridyl, -CO alkyl, -CO aryl, -SO 2 alkyl, -SO 2 aryl, -CO 2 alkyl, C 2 -C 4 (CO) alkenyl , -CO 2 aryl, -SO 3 H;
  • L is hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, the substituent of C 1 -C 10 alkyl is selected from halogen, hydroxyl, -NH 2 , nitro, -CN, C 1 -C 4 alkyl , C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 3 -C 6 cycloalkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl;
  • W 2 is C or NH
  • V 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.
  • Z 1 is hydrogen, substituted or unsubstituted C 1 -C 10 alkyl, the substituent of C 1 -C 10 alkyl is selected from halogen, hydroxyl, -NH 2 , nitro, -CN, C 1 -C 4 alkane C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 3 -C 6 cycloalkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, phenyl, benzyl , pyridyl, -COalkyl, -COaryl, -SO2alkyl , -SO2aryl, -CO2alkyl , C2- C4 ( CO)alkenyl, -CO2aryl , -SO3H ;
  • W 3 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.
  • fragment B in the structural formula of the derivative has the following structure:
  • the derivative is the following compound, the diastereomeric mixture of the following compound or the following compound One of the enantiomers of
  • the derivatives include corresponding deuterated compounds in which any one or more hydrogen atoms are replaced by its stable isotope deuterium.
  • compositions which comprises: the above-mentioned compound of general formula I, its pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug or Its pharmaceutically acceptable carrier.
  • additional therapeutic agents are also included, and the additional therapeutic agents include antidepressants, antimanic drugs, drugs for treating Parkinson's disease, drugs for treating Alzheimer's disease or combinations thereof.
  • the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, triflate, benzenesulfonate, p-toluene Sulfonate (tosylate), 1-naphthalenesulfonate, 2-naphthalenesulfonate, acetate, trifluoroacetate, malate, tartrate, citrate, lactate, oxalate salt, succinate, fumaric acid Salt, maleate, benzoate, salicylate, phenylacetate, mandelate.
  • the additional therapeutic agent is moclobemide, toloxaone, fluoxetine, paroxetine, citalopram, sertraline, venlafaxine, trimipramine, trazodone, Imidamine, desipramine, clomipramine, amitriptyline, nortriptyline, doxepin, maprotiline, loxapine, amoxapine, mirtazapine, buspirone, Clomezadone, tandospirone, lithium carbonate, tacrine, huperzine A, galantamine, donepezil, Lifanstigmine, memantine, pramipexole, tarexol, ropironib, or a combination of them.
  • the present invention also provides the application of the pharmaceutical composition in the preparation of medicines for preventing, treating, treating or alleviating diseases, diseases or conditions of patients.
  • the associated diseases, disorders or conditions caused by mitochondrial abnormalities are specifically associated diseases, disorders or conditions caused by abnormalities in the respiratory chain, including: metabolic diseases, tumors, inflammation, and central nervous system diseases. kind.
  • Metabolic diseases include: hyperglycemia, hyperlipidemia, hypercholesterolemia, high LDL, low HDL, angiogenic disorders, non-alcoholic fatty liver, cerebrovascular accident, myocardial infarction, atherosclerosis , coronary heart disease, anti-aging, urgency and frequent urination, type I diabetes, chronic obstructive pulmonary disease, etc.
  • Tumors include: benign prostatic hyperplasia, Wegener's granulomatosis, pulmonary sarcoidosis, leukemia, lymphoma, pancreatic cancer, neurological tumors, etc.
  • Inflammation includes: peripheral neuritis, chemotherapy-induced peripheral neuritis, autoimmune diseases, conditions associated with organ transplantation, influenza virus, coronavirus (infection prevention, treatment and resolution of sequelae), acute respiratory distress syndrome, inflammatory Enteropathy, Crohn's disease, ulcerative colitis, psoriasis, retinal detachment, retinitis pigmentosa, macular degeneration, pancreatitis, atopic dermatitis, rheumatoid arthritis, spondyloarthritis, gout, systemic lupus erythematosus, Sjogren's syndrome, provincial scleroderma, antiphospholipid syndrome, vasculitis, osteoarthritis, autoimmune hepatitis, autoimmune hepatobiliary disease, primary sclerosing cholangitis, nephritis, chyle Diarrhea, autoimmune ITP, transplant rejection, ischemia-reperfusion injury of solid organs, seps
  • Central nervous system disorders include: Pick's disease, spinal cord injury repair, depression, anxiety, Parkinson's disease, Alzheimer's disease, sleep disorders, ischemic stroke, hemorrhagic stroke, amyotrophic lateral Sclerosis, traumatic brain injury, brain atrophy, Huntington's disease, schizophrenia, mania, drug addiction withdrawal, multiple sclerosis, sleep improvement, muscle weakness, etc.
  • smilagenin-based compounds Using the smilagenin-based compounds, pharmaceutical compositions and applications of the present invention, through relevant in vivo and in vitro model activity tests on the smilagenin-based compounds, it was unexpectedly found that many derivative compounds have superior cytoprotective activity, especially It has unexpected protective activity on a variety of brain neuron cells, and this type of compound has excellent blood-brain permeability, and has potential wide application and great value for the treatment of diseases caused by a variety of mitochondrial dysfunction, making up for The deficiencies of the prior art in the application of timogenin compounds have important scientific and commercial application value.
  • Figure 1 shows the death of neurons under the action of different small molecule compounds.
  • Fig. 2 shows the effect of small molecule compounds on hydrogen peroxide (H 2 O 2 )-induced oxidative damage to human SHSY5Y neural tumor cells.
  • Figure 3 is the percentage of dead neurons.
  • Figure 4 is a phenotype diagram of the effects of each experimental group on zebrafish inflammation.
  • Figure 5 is the effect of each experimental group on zebrafish inflammation (number of neutrophils).
  • Figure 6 shows the anti-inflammatory effects of each experimental group on inflammatory zebrafish.
  • Figure 7 shows changes in cerebral blood flow in mice during the operation.
  • Figure 8 shows the body weight changes of mice before and after surgery.
  • Figure 9 shows the changes in the grip force of the mouse forelimbs.
  • Figure 10 is the score of neurological deficit in mice.
  • Figure 11 shows the volume of cerebral infarction in mice.
  • Figure 12 is the volume of mouse brain edema.
  • Figure 13 is the immobility time of mouse FST.
  • Figure 14 is the preference rate of sugar water in mouse SPT.
  • Figure 15 is the TST immobility time of mice.
  • Figure 16 is the detection of ROS levels in mouse serum.
  • Figure 17 is the detection of H2O2 concentration in mouse serum.
  • Figure 18 is the detection of NO concentration in mouse serum.
  • Figure 19 is the detection of lipid peroxidation level in mouse serum.
  • Figure 20 is the detection of ROS levels in the mouse hippocampus.
  • Figure 21 is the detection of H 2 O 2 concentration in mouse hippocampus.
  • Figure 22 is the detection of NO concentration in mouse hippocampus.
  • Figure 23 is the detection of lipid peroxidation level in mouse hippocampus.
  • Figure 24 is the detection of IL-1 ⁇ concentration in mouse serum.
  • Figure 25 is the detection of IL-6 concentration in mouse serum.
  • Figure 26 is the detection of IL-10 concentration in mouse serum.
  • Figure 27 is the detection of IL-1 ⁇ concentration in mouse hippocampus.
  • Figure 28 is the detection of IL-6 concentration in mouse hippocampus.
  • Figure 29 is the detection of IL-10 concentration in mouse serum.
  • Figures 30a to 32b show the binding strength of each small molecule to Complex I of the respiratory chain, respectively.
  • Figure 33 is a diagram showing the results of the activity of each small molecule on SMP.
  • Figure 34 is a schematic diagram of the results of various small molecules on oxygen consumption of cells.
  • Figures 35a to 35b are graphs showing the results of small molecules on mitochondrial ROS and mitochondrial transmembrane potential difference, respectively.
  • Figures 36a to 36d are graphs showing the effects of small molecules on atherosclerosis in APOE mice, respectively.
  • Figures 37a to 37g are graphs showing the water maze results of small molecules on AD rats.
  • Fig. 38 is a graph showing the results of small molecules on the T-maze of AD rats.
  • Figures 39a and 39b are graphs showing the effect of small molecules on the nesting behavior of AD mice.
  • Figures 40a and 40b are graphs showing the water maze results of small molecules on AD mice.
  • Figure 41 is a graph showing the light and dark box results of small molecules on AD mice.
  • Figures 42a and 42b are graphs of small molecule effects on survival of TDP43 A315T mice.
  • Fig. 43 is a graph showing the results of small molecule analysis on the gait of SOD G93A mice.
  • Figure 44 is a graph of the open field results of small molecules on SOD G93A mice.
  • Figures 45a and 45b are graphs showing the results of small molecules on the DSS mouse enteritis model.
  • Figures 46a and 46b are graphs showing the results of small molecules on the TNBS rat enteritis model.
  • Figure 47 is a graph showing the effect of small molecules on blood glucose in DB mice.
  • Figures 48a to 48d are the result graphs of body weight and body fat percentage of DIO mice.
  • Figure 49 is a graph showing the in vivo killing effect of small molecules on hematological tumors.
  • Fig. 50 is a result graph showing the protective effect of small molecules on substantia nigra neurons.
  • Figure 51 is a graph showing the results of the protective effect of small molecules on virus infection.
  • Fig. 52 is a graph showing the results of small molecules on the acute craniocerebral trauma model of rat TBI.
  • Fig. 53 is a graph showing the in vitro killing effect of small molecules on pancreatic cancer cells.
  • Figure 54 is a graph of the results of small molecule excess urine excretion in DB animals.
  • Figures 55a to 55c are graphs of small molecule effects on cardiovascular inflammation in high fat fed APOE mutant mice.
  • Figures 56a and 56b are graphs showing the experimental results of the effect of small molecules on the sleep of mice affected by a subthreshold hypnotic dose of pentobarbital sodium.
  • alkyl in the present invention refers to a monovalent saturated aliphatic hydrocarbon group with 1 to 10 carbon atoms, including straight chain and branched chain hydrocarbon groups, such as methyl (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CH 3 ) 2 CH-), n-butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl (( CH 3 ) 2 CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), tert-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH 2 CH 2 CH 2 —), neopentyl ((CH 3 ) 3 CCH 2 —).
  • alkyl includes substituted or unsubstituted alkyl groups.
  • substituted or unsubstituted means that the group can be unsubstituted, or the H in the group can be replaced by one or more (preferably 1 to 6, more preferably 1 ⁇ 3) substituents.
  • the "substituted" means that the group has one or more (preferably 1-6, more preferably 1-3) substituents selected from the following group: halogen, hydroxyl , -NH 2 , nitro, -CN, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 3 -C 6 cycloalkyl, C 2 -C 4 Alkenyl, C 2 -C 4 alkynyl, phenyl, benzyl, C 2 -C 8 heterocyclyl, C 2 -C 8 heteroaryl, heteroatoms are selected from one or more of N, O and S indivual.
  • substituents selected from the following group: halogen, hydroxyl , -NH 2 , nitro, -CN, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, C 1 -C 4 alkoxy, C 3 -C 6 cycloalky
  • cycloalkyl represents a substituted or unsubstituted C 3 -C 12 cycloalkyl.
  • alkoxy refers to an -O-alkyl group, wherein the alkyl group may be saturated or unsaturated, branched, linear, or cyclic.
  • 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, propoxy.
  • aryl refers to a monovalent aromatic carbocyclic group of 6 to 20 (preferably 6 to 14) carbon atoms, which has a single ring (such as phenyl) or a condensed ring (such as naphthalene) group or anthracenyl), if the point of attachment is on an aromatic carbon, the fused ring may be non-aromatic (such as 2-benzoxazolone, 2H-1,4-benzoxazin-3(4H)-one- 7-base, etc.).
  • Preferred aryl groups include phenyl and naphthyl.
  • the term includes substituted or unsubstituted forms wherein the substituents are as defined above.
  • alkenyl groups are vinyl, allyl, but-3-enyl.
  • cycloalkyl refers to a cyclic alkyl group having 3 to 10 carbon atoms, having a single ring or multiple rings (including fused systems, bridged ring systems and spiro ring systems). In fused ring systems, one or more rings can be cycloalkyl, heterocyclic, aryl, or heteroaryl as long as the point of attachment is through the cycloalkyl ring.
  • suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclooctyl.
  • halo or halogen refers to fluorine, chlorine, bromine and iodine.
  • heteroaryl refers to an aromatic group having 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur in the ring, such a heteroaryl group may be monocyclic (such as pyridyl or furyl) or fused ring (such as indolizinyl (indolizinyl) or benzothienyl), wherein the fused ring can be non-aromatic and/or contain a heteroatom, as long as the point of attachment is through an atom of an aromatic heteroaryl.
  • the ring atom nitrogen and/or sulfur of the heteroaryl is optionally oxidized to N-oxide (N-O), sulfinyl or sulfonyl.
  • N-O N-oxide
  • Preferred heteroaryl groups include pyridyl, pyrrolyl, indolyl, thienyl and furyl. The term includes substituted or unsubstituted heteroaryl groups.
  • substituted heteroaryl refers to a heteroaryl group substituted by 1 to 5, preferably 1 to 3, more preferably 1 to 2 substituents selected from and The same substituents as defined for substituted aryl.
  • heterocycle or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl” refers to a saturated, partially saturated or unsaturated group (but not aromatic) , having a single ring or a condensed ring (including a bridged ring system and a spiro ring system), with 1 to 10 carbon atoms and 1 to 4 (such as 3) heteroatoms selected from nitrogen, sulfur or oxygen in the ring, in the fused In a ring system, one or more rings may be cycloalkyl, aryl or heteroaryl as long as the point of attachment is through a non-aromatic ring.
  • the nitrogen and/or sulfur atoms of the heterocyclic group are optionally oxidized to provide N-oxide, sulfinyl and sulfonyl moieties.
  • substituted heterocyclic or “substituted heterocycloalkyl” or “substituted heterocyclyl” refers to a heterocyclic ring substituted by 1 to 5 (eg 1 to 3) substituents group, the substituents are the same as defined for substituted cycloalkyl.
  • stereoisomer refers to compounds that differ in chirality at one or more stereocenters.
  • Stereotropic Conomers include enantiomers and diastereomers.
  • the term "tautomer” refers to alternative forms of compounds that differ in the position of the proton, such as enol-keto and imine-enamine tautomers, or tautomers of heteroaryl groups
  • the heteroaryl group contains ring atoms attached to the -NH- part of the ring and the N- part of the ring, such as pyrazole, imidazole, benzimidazole, triazole and tetrazole.
  • the invention provides a pharmaceutical composition, which contains active ingredients in a safe and effective dose range, and a pharmaceutically acceptable carrier.
  • the "active ingredient" in the present invention refers to the compound of general formula (I) or its pharmaceutically acceptable salt, its stereoisomer or its tautomer, or its prodrug in this invention.
  • the "active ingredients" and pharmaceutical compositions described in the present invention can be used as mitochondrial protective agents. In another preferred embodiment, it is used for preparing medicines for preventing and/or treating neurodegenerative diseases. In another preferred embodiment, it is used for preparing medicines for preventing and/or treating metabolic diseases related to mitochondria.
  • Safe and effective amount means: the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg active ingredient/dose, more preferably 10-200 mg active ingredient/dose.
  • the "one dose” is a tablet.
  • “Pharmaceutically acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and low toxicity. "Compatibility” here means that each component in the composition can be blended with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient.
  • the compounds of the preferred embodiments of the present invention may be administered as the sole active agent or in combination with one or more other agents useful in the treatment of cancer.
  • the compounds of the preferred embodiments will be administered in a therapeutically effective amount by any of the accepted modes of agents having similar effects.
  • the actual amount of the compound (i.e., active ingredient) of the preferred embodiments to be used will depend on a number of factors, such as the severity of the disease being treated, the age and relative health of the patient, the potency of the compound being used, the route and form of administration, and other factors. .
  • the drug can be administered several times a day, preferably once or twice a day. All of these factors are within the consideration of the attending physician.
  • the therapeutically effective dose can generally be a total daily dose administered to the patient once or in divided doses, 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.
  • Dosage unit compositions may contain dosage factors thereof to yield the daily dose. The choice of dosage form depends on various factors, such as the mode of administration and the bioavailability of the drug substance.
  • the compounds of the preferred embodiments may be administered as pharmaceutical compositions by any route appropriate for the condition being treated. Suitable routes include, but are not limited to, oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intradermal), vaginal, intraperitoneal, intrapulmonary and intranasal.
  • the preferred route may vary depending on the condition of the patient.
  • the preferred mode of administration is oral administration, and the convenient daily dose can be adjusted according to the degree of bitterness. It can be formulated with pharmaceutically acceptable carriers or excipients as tablets, pills, capsules, semi-solids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other suitable composition wait.
  • the compound When the compound is formulated parenterally, it can be formulated with a pharmaceutically acceptable parenteral carrier.
  • Another preferred mode of administering the compounds of the preferred embodiments is by inhalation. This is an effective method of delivering therapeutic agents directly to the respiratory tract (see, eg, US Patent No. 5,607,915).
  • the present invention can administer the compound in any convenient preparation form, and the "preparation" referred to in the present invention refers to a dosage form containing the compound of general formula I of the present invention that is beneficial to administration (drug delivery), such as: but not limited to, aqueous solution injection, Powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder sprays, sustained-release and controlled-release preparations, etc.
  • drug delivery such as: but not limited to, aqueous solution injection, Powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder sprays, sustained-release and controlled-release preparations, etc.
  • These pharmaceutical excipients can be commonly used in various preparations, such as: but not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants and lubricants, etc. ; It can also be selected for use in order to be compatible with the substance, such as: emulsifier, solubilizer, bacteriostat, analgesic and antioxidant, etc., this type of adjuvant can effectively improve the stability of the compound contained in the composition and solubility or change the release rate and absorption rate of the compound, etc., thereby improving the metabolism of the compound of the present invention in vivo, thereby enhancing the administration effect.
  • excipients used to achieve specific administration purposes or methods such as sustained-release administration, controlled-release administration, and pulse administration, such as but not limited to, Gelatin, albumin, chitosan, polyether and polyester polymer materials, such as: but not limited to, polyethylene glycol, polyurethane, polycarbonate and its copolymers, etc.
  • the main manifestations of the so-called “beneficial” include, but are not limited to, improving the therapeutic effect, increasing bioavailability, reducing toxic and side effects, and improving patient compliance.
  • Suitable pharmaceutically acceptable carriers or excipients include, for example, treating agents and drug delivery modifiers and enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose , methylcellulose, sodium carboxymethylcellulose, glucose, hydroxypropyl- ⁇ -cyclodextrin, sodium sulfobutyl- ⁇ -cyclodextrin, polyvinylpyrrolidone, low melting point wax, ion exchange resin, etc., and Any combination of two or more thereof.
  • treating agents and drug delivery modifiers and enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose , methylcellulose, sodium carboxymethylcellulose, glucose, hydroxypropyl- ⁇ -cyclodextrin, sodium sulfobutyl- ⁇ -cyclodextrin, polyvinylpyrrolidone, low melting point
  • Liquid and semisolid excipients can be selected from glycerol, propylene glycol, water, ethanol and various oils, including petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Preferred liquid carriers, especially for injectable solutions include water, saline, aqueous dextrose and glycol.
  • Other suitable pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991), incorporated herein by reference.
  • the term "pharmaceutically acceptable salt” refers to non-toxic acid or alkaline earth metal salts of compounds of general formula I. These salts can be prepared in situ during the final isolation and purification of the 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, lauryl sulfate, ethanesulfonate, glucose heptanoate, glycerophosphate, hemisulfate, heptanoic acid Salt, Caproate, Fumarate, Hydrochloride, Hydrobromide, Hydroiodide, 2-Hydroxyethanesulfonate, Lactate, Maleate, Methanesulfonate, Nicotinate , 2-naphthylsulfonate, oxalate, pamoate, pectate, thiocyanate, 3-phenylpropionate, picrate, pivalate, propionate, Succinate, Sulfate
  • nitrogen-containing basic groups can be quaternized with the following reagents: alkyl halides, such as chlorides, bromides, and iodides of methyl, ethyl, propyl, and butyl groups; dialkyl sulfates , such as dimethyl, diethyl, dibutyl, and dipentyl sulfate; long-chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; arane Halides such as benzyl and phenethyl bromide, etc. Water-soluble or oil-soluble or dispersible products are thus obtained.
  • alkyl halides such as chlorides, bromides, and iodides of methyl, ethyl, propyl, and butyl groups
  • dialkyl sulfates such as dimethyl, diethyl, dibutyl, and dipentyl s
  • acids which can be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and organic acids such as oxalic acid, maleic acid, methanesulfonic acid, succinic acid, citric acid.
  • Base addition salts can be prepared in situ during the final isolation and purification of the compounds of general formula I, or by separately reacting the carboxylic acid moiety with a suitable base such as a pharmaceutically acceptable metal cation hydroxide, carbonate or carbonic acid Hydrogen salt) or ammonia, or organic primary, secondary or tertiary amine reaction.
  • Pharmaceutically acceptable salts include, but are not limited to, alkali metal and alkaline earth metal based cations, such as sodium, lithium, potassium, calcium, magnesium, aluminum salts, etc., as well as non-toxic ammonium, quaternary ammonium and amine cations, including , but not limited to: ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc.
  • Other representative organic amines for the formation of base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like.
  • the term "pharmaceutically acceptable prodrug” refers to those prodrugs of the compounds of the preferred embodiments, which are rapidly converted into the parent compound represented by the above general formula in vivo, for example, hydrolyzed in blood.
  • pharmaceutically acceptable prodrug refers to those prodrugs of the compounds of the preferred embodiments, which are rapidly converted into the parent compound represented by the above general formula in vivo, for example, hydrolyzed in blood.
  • T. Higuchi and V. Stella Pro-drugs as Novel Delivery Systems (Pro-drugs as Novel Delivery Systems), Volume 14 of A.C.S.15 Symposium Series” and “Edward B. Roche, eds., Bioreversible Carriers in Drug Design (Bioreversible Carriers in Drug Design), American Pharmaceutical Association and Pergamon Press, 1987", both of which are incorporated herein by reference.
  • the present invention provides the preparation method of the compound of general formula (I). Taking timogenin as an example, the preparation method of the key intermediate is as follows:
  • the preparation of the compound of general formula (I) is as follows: Take the timosaponin mother nucleus ⁇ configuration as an example (other configurations or certain configurations of other mother nucleus, the preparation method is consistent with the method provided, and the synthetic route is as follows:
  • R 1 , R 2 , Y, and n are as above.
  • DBU refers to 1,8-diazabicyclo[5.4.0]undec-7-ene
  • DIBAL refers to diisobutylaluminum hydride
  • DIAD refers to diisopropyl azodicarboxylate
  • DIEA refers to diisopropyl Ethylamine
  • 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 means dimethylsulfoxide
  • DPPB means 1,4-bis(diphenylphosphino)butane
  • DPPE means 1,2-bis(diphenylphosphino)ethane
  • DPPF means 1,1'-bis(diphenylphosphino)ferrocene
  • DPPM means 1,1'-bis(dipheny
  • the preparation process is as follows:
  • intermediate 8 was obtained under the same conditions as in the third step in Example 1A, with a yield of 50%.
  • intermediate 9 was obtained under the same conditions as in the fourth step in Example 1A, with a yield of 90%.
  • Example 1B was obtained, and the yield 65%.
  • Example 2 was obtained through the same experimental conditions as described in the fourth step in Example 1.
  • Example 3 was obtained through the same experimental conditions as described in the fourth step in Example 1.
  • Example 4 was obtained through the same experimental conditions as described in the fourth step in Example 1.
  • Example 4A was obtained through the same experimental conditions as described in the fifth step in Example 1A.
  • Example 4B was obtained through the same experimental conditions as described in the fifth step in Example 1A.
  • Example 5 was obtained through the same experimental conditions as described in the fourth step in Example 1.
  • Example 6 was obtained through the same experimental conditions as described in the fourth step in Example 1.
  • Example 8 was obtained through the same experimental conditions as described in the fourth step in Example 1.
  • Example 9 was obtained through the same experimental conditions as described in the fourth step of Example 1.
  • Example 10 was obtained through the same experimental conditions as described in the fourth step in Example 1.
  • intermediate 12 In a 500 mL round bottom flask were charged intermediate 12 (5 g, 1 eq), mono-tert-butyl malonate (2 eq), EDC ⁇ HCl (2 eq), DMAP (0.1 eq) and Et3N (5 eq), Dissolve in 100 ml of dry DCM, stir the reaction at room temperature for 2 h, and follow the end of the reaction by TLC (PMA color development). After the reaction was completed, the product was washed twice with saturated NH4Cl aqueous solution, dried, and subjected to silica gel column chromatography to obtain 4.5 g of the product.
  • NMR data are as follows: 1HNMR (CDCl3, 400MHz, ppm): ⁇ 0.76 (s, 3H), ⁇ 0.80-2.30 (m, 36H), ⁇ 2.55-3.00 (m, 9H), ⁇ 3.15-4.50 ( m, 14H), ⁇ 5.25 (br s, 1H); mass spectrum: [M+1] 641.5.
  • reaction solution After the reaction is completed, slowly drop the reaction solution into water (65L) at 0-10°C, a large amount of solids precipitate out, filter, and rinse the filter cake with 500mL of PE: EA (50:1) mixed solvent, and the obtained solids are vacuum-washed. Dry at low temperature (40-50° C. water bath) for 8 hours to obtain 451 g of solid, namely intermediate 13.
  • intermediate 13 25 g, 43.8 mmol
  • potassium acetate 8.60 g, 87.6 mmol
  • 18-crown-6 23.15 g, 87.6 mmol
  • 300 mL of dimethylsulfoxide solvent 300 mL
  • the temperature of the reaction system was raised to 55 degrees Celsius, and the temperature was maintained for 16 hours.
  • the reaction mixture was poured into 1 L of ice water and stirred for 30 minutes. Filter and wash the filter cake with water to give 7 g of white solid.
  • intermediate 14 (2.0 g, 4.36 mmol) was added along with 60 mL THF, 60 mL methanol, 30 mL water and 5.5 mL 4N aqueous LiOH.
  • the temperature of the reaction system was raised to 60 degrees Celsius, and the temperature was maintained for 2 hours.
  • the organic solvent was spun off, and 50 ml of water was added. Filtration and washing of the filter cake with water yielded 1.7 g of a white solid.
  • TMSN 3 (0.12g, 1.1eq, 2eq) and DBU (0.32g, 0.21mmol, 4eq ), N 2 .
  • the reaction solution was introduced into water (20 mL) at 0-10°C, stirred at 0-10°C for 5-10 minutes, and then filtered. The filter cake was purified by silica gel column chromatography and eluted with PE:EA to obtain 0.13 g of intermediate 16 as a solid, with a yield of 56%.
  • DCM/MeOH 5% ammonia methanol
  • Example 14 Using intermediate 18, the same synthesis method as Example 14 can be used to obtain white solid Example 14A.
  • Example 14B the white solid Example 14B can be obtained by the same synthesis method as Example 14.
  • Example 15 was obtained by the same method as Example 14.
  • Example 15A was obtained by the same method as Example 14A.
  • Example 15B was obtained by the same method as Example 14A.
  • Example 16A was obtained by the same method as Example 13A.
  • Example 16B was obtained through the same preparation steps and conditions as Example 13B.
  • Example 17A was obtained.
  • Example 18A was obtained through the same preparation steps and conditions as described in Example 4A.
  • Example 18B was obtained through the same preparation steps and conditions as those described in Example 4B.
  • Example 19A Taking heikegenin as a raw material, through the same preparation steps and conditions as intermediate 7, intermediate 24 was obtained; using intermediate 24 as a raw material, through the same preparation method and conditions as described in step 5 of Example 1A, to obtain Example 19A.
  • Example 20A was obtained by using hecogenin as a raw material through the same preparation steps and conditions as those described in Example 4A.
  • Example 21 was obtained through the same preparation steps and conditions as described in Example 13.
  • Example 22 was obtained through the same preparation steps and conditions as described in Example 13.
  • Example 24A Example 24B
  • Example 24A can be obtained by the same synthesis method as Example 24.
  • Example 24B was obtained in the same way.
  • the nuclear magnetic data of embodiment 24A is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 24B nuclear magnetic data is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 25A Example 25B
  • Example 25A can be obtained by the same synthesis method as Example 24.
  • Example 25B was obtained in the same way.
  • the nuclear magnetic data of embodiment 25A is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • the nuclear magnetic data of embodiment 25B is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 26A can be obtained by the same synthesis method as Example 24.
  • Example 26B was obtained in the same way.
  • the nuclear magnetic data of embodiment 26A is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Embodiment 26B magnetic data is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 27A Example 27B
  • Example 27A can be obtained by the same synthesis method as Example 27.
  • Example 27B was obtained in the same way as intermediate 21 and N-methyl-2-(1-methylpiperidin-4-yl)ethan-1-amine.
  • the nuclear magnetic data of embodiment 27A is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • the nuclear magnetic data of embodiment 27B is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 29A Example 29B
  • Example 29A The synthesis process is the same as that of Example 29, and Example 29A can be obtained by using the same synthesis method as Example 29 with Intermediate 18 and 1-(1-ethylpiperidin-4-yl)piperazine. Using intermediate 21 and 1-(1-ethylpiperidin-4-yl)piperazine, Example 29B was obtained in the same way.
  • the nuclear magnetic data of embodiment 29A is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • the nuclear magnetic data of embodiment 29B is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 32A Example 32B
  • Example 32A can be obtained by the same synthesis method as Example 32.
  • Example 32B was obtained in the same way.
  • the nuclear magnetic data of embodiment 32A is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 32B The NMR data of Example 32B are: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 33A Example 33B
  • Example 33A can be obtained by the same synthesis method as Example 33.
  • Example 33B was obtained in the same way.
  • Example 33B nuclear magnetic data is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 34A Example 34B
  • Example 34A can be obtained by the same synthesis method as Example 34.
  • Example 34B was obtained in the same way.
  • the nuclear magnetic data of embodiment 34A is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 34B The NMR data of Example 34B are: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 35 Using intermediate 11 and 1-(pyridin-4-yl)piperazine as raw materials, the same synthesis method as in Example 28 can be used to obtain Example 35
  • Example 35A Example 35B
  • Example 35A can be obtained by the same synthesis method as Example 28.
  • Example 35B was obtained in the same way.
  • the nuclear magnetic data of embodiment 35A is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • the nuclear magnetic data of embodiment 35B is: 1H NMR (400MHz, CDCl3) ⁇ 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.
  • Example 36 can be obtained by the same synthesis method as Example 31
  • Example 41A Example 41B
  • Example 41A The synthesis process of Example 41A is as in Example 41, Intermediate 18 is replaced by Intermediate 11, and the nuclear magnetic data is 1H NMR (CDCl3-d6, 400MHz): 7.98 (d, 1H), 4.42 (q, 1H), 4.18 ( s,1H),3.95(dd,1H),3.66(t,2H),3.57(t,2H),3.31(d,1H),3.29(s,2H),2.67-2.82(m,8H),2.47 -2.52(m,4H),1.58-2.10(m,11H),1.15-1.50(m,16H),1.09-1.10(m,6H),1.08(d,3H),0.99(d,3H),0.95 (s,3H), 0.75(s,3H). Mass spectrum: [M+1] 669.7.
  • mice 15-day-pregnant rats, dissect fetal mouse brains (E15-16), use 48-well plates for primary culture of cerebral cortex neurons in neuron medium for 14 days (DIV 14), and perform OGD experiment.
  • the neuron culture medium was replaced with a sugar-free anaerobic culture medium (95% N 2 /5% CO 2 balance), and the neurons were treated with OGD in the OGD chamber. Then it was replaced with normal neuron culture medium, cultured in 95% Air/5% CO 2 incubator for 24 hours, and then the neuron activity was analyzed.
  • Compound treatment Dilute the 13 small molecule compounds from Examples 1 to 20 to 0.5mM mother solution with DMSO, add 1 ⁇ L of the mother solution to 0.5ml culture solution in each well of a 48-well plate to a final concentration of 1 ⁇ M, and 1 ⁇ L of the positive control compound DPQ (mother solution 10mM), negative control 1 ⁇ L DMSO was added to the culture medium 24 hours before the OGD experiment, and the same concentration was added to the sugar-free and anaerobic culture medium and the subsequent normal neuron culture medium.
  • Example 2 As shown in Figure 2, Example 2, Example 7, Example 13, and Example 14A significantly protected neurotumor cell damage induced by hydrogen peroxide, wherein, undifferentiated SH-SY5Y cells were pre-protected for 24 hours, and finally The concentration was 1 ⁇ M, and it was detected after 24 hours of hydrogen peroxide treatment.
  • the 24-well cell culture plate was pretreated with Poly-D-lysine, and placed in a cell culture incubator at 37°C and 5% CO 2 overnight.
  • test compound final concentration 1 ⁇ M
  • positive control AP5 [(2R)-amino-5-phosphovaleric acid (ester)] (final concentration 100 ⁇ M)
  • L-Cystine 400 ⁇ M
  • NaHCO 3 10 mM
  • Compound treatment Dilute the 32 small molecule compounds from Examples 1 to 19A to 1 mM mother solution with DMSO, add 1 ⁇ l of the mother solution to 1 ml of the culture solution in each well of the 24-well plate to a final concentration of 1 ⁇ M, and 100 ⁇ l of the positive control compound AP5 ( stock solution 100mM), negative control 1 ⁇ l DMSO.
  • Transgenic neutrophil-fluorescing zebrafish were reproduced by natural pair mating. The age is 3 days after fertilization (3dpf), a total of 810 tails, 30 tails in each experimental group. It is used to determine the maximum detection concentration (MTC) of "Example 5" and “Example 6" in the LPS-induced inflammation experiment and the anti-inflammatory effect evaluation of "Example 5" and "Example 6" on LPS-induced inflammation.
  • MTC maximum detection concentration
  • Zebrafish are kept in 28°C fish farming water (water quality: add 200mg of instant sea salt to every 1L of reverse osmosis water, conductivity is 480-510 ⁇ S/cm; pH is 6.9-7.2; hardness is 53.7-71.6mg/L CaCO 3 ) , provided by the fish breeding center of the company, the license number for the use of experimental animals is: SYXK (Zhejiang) 2012-0171.
  • the feeding management complies with the requirements of the international AAALAC certification.
  • Example 5 white powder, stored dry at 4°C, received on October 18, 2019, provided by Shenzhen Qingbo Huineng Pharmaceutical Technology Co., Ltd. Prepare 20mM mother solution with DMSO before the experiment and store at -20°C.
  • Example 6 white powder, stored dry at 4°C, received on October 18, 2019, provided by Shenzhen Qingbo Huineng Pharmaceutical Technology Co., Ltd. Prepare 20mM mother solution with DMSO before the experiment and store at -20°C.
  • 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
  • Dimethylsulfoxide Sigma, France
  • 6-well plate Nest Biotech
  • LPS was injected into the yolk sac to treat normal 3dpf transgenic neutrophil fluorescent zebrafish to establish a zebrafish inflammation model.
  • zebrafish treated with water for fish farming and model The control group was placed in an incubator at 28°C for 3 hours to observe and record the death of zebrafish, count the number of zebrafish deaths in each experimental group, and determine the maximum detection of zebrafish in "Example 5" and "Example 6" concentration (MTC).
  • Inflammation regression effect (%) ((N (model control group)-N (test product group))/N (model control group))*100%
  • the maximum solubility of "Example 5" in DMSO is 20mM, and the maximum concentration of DMSO that zebrafish can tolerate is 1%, so the maximum detection concentration of "Example 5" anti-inflammatory effect evaluation is 200 ⁇ M, "Example 5" is in At 200, 100, 50 and 10 ⁇ M concentrations, 30 zebrafish died, and the mortality rate was 100%; at 5 ⁇ M concentration, 3 zebrafish died, and the mortality rate was 10%; Therefore, the maximum detection concentration for the evaluation of the anti-inflammatory effect of "Example 5" was 2.5 ⁇ M.
  • Example 6 has a maximum solubility in DMSO of 20 mM, and the maximum concentration of DMSO that zebrafish can tolerate is 1%, so the maximum concentration of "Example 6" anti-inflammatory evaluation is 200 ⁇ M, "Example 6" At the concentration of 200, 100 and 50 ⁇ M, 30 zebrafish died, and the mortality rate was 100%; at the concentration of 10 and 5 ⁇ M, 4 zebrafish died, and the mortality rate was 13.33%; at the concentration of 2.5 ⁇ M, the state of the zebrafish was normal, The drug was not precipitated, so the maximum detection concentration for the evaluation of the anti-inflammatory effect of "Example 6" was 2.5 ⁇ M.
  • Figure 4 the dotted line area in Figure 4 is the neutrophils at the site of inflammation.
  • the number of neutrophils in the inflammatory site of the model control group (18) was compared with the normal control group (3), p ⁇ 0.001, indicating that the LPS-induced transgenic neutrophil fluorescence zebrafish inflammation model was successfully established.
  • the number of neutrophils in the inflammatory site of 80 ⁇ M indomethacin group (6) was less than 0.001, and its anti-inflammatory effect was 67%, indicating that indomethacin has obvious anti-inflammatory effects on inflammatory zebrafish effect.
  • Example 5 When the concentration of "Example 5" was 0.28, 0.83, and 2.5 ⁇ M, the number of neutrophils in the zebrafish inflammatory site was 12, 7, and 6, respectively, and the anti-inflammatory effects on zebrafish were 33%, 61%, and 67%; compared with the model control group (18), p ⁇ 0.001 in the 0.28, 0.83 and 2.5 ⁇ M concentration groups, suggesting that "compound 3" has obvious anti-inflammatory effects on inflammatory zebrafish at a concentration of 0.28-2.5 ⁇ M.
  • Example 6 when the concentration is 0.28, 0.83, 2.5 ⁇ M, the number of neutrophils in the zebrafish inflammation site is 12, 8 and 7 respectively, and the anti-inflammatory effect on zebrafish is respectively 33%, 56% and 61%; compared with the model control group (18), p ⁇ 0.001 in the 0.28, 0.83 and 2.5 ⁇ M concentration groups, suggesting that "compound 4" has obvious anti-inflammatory effect on inflammatory zebrafish at the concentration of 0.28-2.5 ⁇ M.
  • Example 5 Under the concentration conditions of this experiment, both Example 5 and Example 6 have obvious anti-inflammatory effects on inflammatory zebrafish.
  • Neurological function scores and cerebral infarct size were used to evaluate the effect of compounds on the mouse cerebral ischemia-reperfusion (MCAO) injury model. The results show that the test compound has the effect of protecting mice from cerebral ischemia-reperfusion (MCAO) injury.
  • Physiological saline Name Physiological saline homemade.
  • Hydroxypropyl- ⁇ -cyclodextrin Name: Hydroxypropyl- ⁇ -cyclodextrin; Provider: Sarn Chemical Technology (Shanghai) Co., Ltd.; Batch: FG310174; Properties: White solid powder; Quantity: 30g; Storage conditions: room temperature.
  • Experimental animals Strain C57BL/6 mice; Week age: 6-8; Sex: male; Order animal weight: 16-20g; Use animal weight: 20-23g; Quantity: 40; Experimental animal provider: Zhejiang Weitongli Hua Experimental Animal Technology Co., Ltd.; production license number: SCKX (Zhejiang) 2019-0001; quality certificate number: No2005130052, No2004280010.
  • Quarantine The quarantine period is 7 days. Routine health checks are completed by veterinarians. Animals with abnormal performance are removed before the experiment.
  • Animal feeding conditions The experimental animals were kept in an SPF-grade laminar flow clean room with constant temperature and humidity in the Animal Center (AAALAC certification unit), with 3 mice per cage.
  • the temperature of the breeding room is 22 ⁇ 3°C, the humidity is 40-70%, and the light is alternated between light and dark for 12 hours.
  • Cage Made of polycarbonate.
  • Feed and drinking water Clean grade mouse feed was purchased from Beijing Keao Xieli Feed Co., Ltd. Drinking water is autoclaved and food is irradiated with cobalt-60 rays. Animals had free access to sterile food and water.
  • Animal number Each cage has a cage label, indicating the number of animals, sex, strain, receiving time, group and the start time of the experiment. Animal number: Each animal is marked with an individual animal number on the tail.
  • the specific grouping and treatment are shown in Table 6.
  • mice were fasted overnight before surgery, but not water.
  • the mice were pre-anesthetized in an induction box of an isoflurane gas anesthesia machine with a concentration of isoflurane of 2.5%. After clamping the unresponsive hind paw of the mouse with dissecting forceps, it was transferred to an anesthesia mask and the concentration of isoflurane was reduced to 1.5%.
  • the body temperature of the mice during the operation was maintained at around 37°C by using a body temperature maintenance instrument and a rectal temperature probe.
  • the hair on the neck of the mouse in the supine position was shaved, and the skin was disinfected with povidone iodine and alcohol, and an incision was made in the middle of the neck.
  • the tissue was bluntly dissected, and the common carotid artery (CCA) was exposed under a stereomicroscope, and its proximal end was ligated with a 6-0 braided suture.
  • CCA common carotid artery
  • ICA internal carotid artery
  • the ECA was fused with a coagulation pen, the external carotid artery ligation was loosened, and the thread plug was inserted into the ICA until the cerebral blood flow stopped at about 10% of the baseline.
  • the thread plug blocked the middle cerebral artery (MCA) for 30 minutes. After 30 minutes, the thread plug was pulled out, the stump of the vessel was cauterized, and the CCA ligation was loosened.
  • the skin of the neck was sutured, and the mice were placed in an intensive care cage, and the body temperature of the mice was maintained at 37°C until the materials were collected.
  • Cerebral blood flow measurement Fix the head of the mouse under the stereotaxic instrument, shave the hair of the head of the mouse, and make a median incision. The periosteum on the skull was removed, and the optical fiber of the laser Doppler flowmeter was fixed with glue at the coordinates of Bregma AP 1.0mm, ML5.0mm, and the blood flow changes during the operation of the middle cerebral artery were recorded. The successful standard of modeling is that the cerebral blood flow drops to 80%-90% of the baseline.
  • Preparation of PK samples First, calculate at least the required drug dose m2 per day based on the total weight of each group of mice and the dosage. Weigh an amount of P2 slightly more than m2 into a bottle, and calculate the solvent volume v2 according to the administration volume of 10mL/kg. Add 20% hydroxypropyl- ⁇ -cyclodextrin solution (v2) to the vial, vortex for one minute, sonicate for 15 minutes, and vortex for another minute until dissolved.
  • v2 hydroxypropyl- ⁇ -cyclodextrin solution
  • Preparation of PK sample Firstly, according to the total weight of each group of mice and the dosage, calculate at least the required dosage m3 per day. Weigh a little more than m3 of P3 into a mortar, and calculate the solvent volume v3 according to the administration volume of 10mL/kg, and measure the solvent of v2 with a syringe. Add 2-3 drops of solvent to the mortar, grind for 5 minutes, after the solvent is dry, add 2-3 drops of solvent, and grind for 5 minutes, repeat three times. Then, wash the compound in the mortar several times with solvent and add it to a suitable sample bottle with a clean glass dropper, sonicate for 15 minutes, and vortex for five minutes until it is uniform and there are no larger particles.
  • Body weight Record the body weight of mice on the day before operation and 24 hours after operation.
  • Neurological function score Longa behavioral score was performed 2h and 24h after the operation of the mice.
  • mice 24 hours before the operation and 24 hours after the operation, the forelimb grip test was performed on the mice respectively.
  • the mice were placed on the grip meter, and the mice actively pulled the grip sensor rod, and the peak value of the grip force was recorded, and the average value was obtained by repeating the measurement three times.
  • mice 24 hours after the operation, the mice were anesthetized with isoflurane and sacrificed by decapitation.
  • the whole brain was peeled off, washed twice with normal saline, placed under the coronal brain mold, the anterior 1mm olfactory bulb and the posterior 4mm cerebellum were removed, and the brain was cut into 2mm pieces, a total of 4 pieces.
  • the volume of cerebral infarction and edema were measured by software ImageJ.
  • Graghpad Prism 7.0 was used for data statistics in each group, and the experimental results were expressed as "mean ⁇ standard deviation”. Statistical method One-way analysis of variance was used to compare whether there was a statistical difference among the groups, and P ⁇ 0.05 was considered statistically significant.
  • the cerebral blood flow of each group dropped to about 10% of the baseline, indicating that the model is successful and can be evaluated for drug efficacy.
  • the neurological deficit scores of the 2h MCAO model group after surgery were significantly different from those of the No. 2 administration group and the No. 3 administration group (P ⁇ 0.05); the neurological deficit scores of the 24h MCAO model group after surgery There were significant differences (P ⁇ 0.05) between the score and the positive control drug edaravone group and the No. 3 administration group respectively.
  • the results of TTC staining showed that the volume of cerebral infarction in the MCAO model group was significantly different from that of 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) have significant difference.
  • the results of TTC staining showed that the volume of brain edema in the MCAO model group was significantly higher than that of 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) had significant difference.
  • Example 6 has an antidepressant effect on mice.
  • NC Solvent Sodium carboxymethyl cellulose
  • Provider Merrill
  • Batch 69881020
  • Properties white solid powder
  • Quantity 200g
  • Storage conditions room temperature
  • LPS Peripheral and central inflammation inducer
  • Experimental animal strain C57BL/6J mice; age: 6-8 weeks; sex: male; animal weight: 18-20g; quantity: 24; experimental animal provider: Guangdong Medical Experimental Animal Center
  • Quarantine The quarantine period is 5 days, and animals with abnormal performance are removed before the experiment.
  • Animal number each cage has a cage label, indicating the number of animals, sex, strain, receiving time, group and the start time of the experiment.
  • Animal number Each animal is marked with an individual animal number on the tail.
  • LPS (2mg/kg, ip, QD) was injected on the 1st, 2nd, and 3rd day to induce peripheral and central inflammation in mice.
  • the ND drug test group was given pre-protection (ND 30mg/kg, po, QD) 2 hours before LPS injection, and forced swimming, tail suspension and sugar water preference experiments were carried out in experiment D4.
  • Solvent (0.5% CMC-Na solution): Take 0.5g CMC-Na white solid powder, add 100ml double distilled water, vortex until dissolved. The prepared solution should be stored in a 4-degree refrigerator and sealed. The solution can be stored for one month, but if it is found to be moldy, it is forbidden to be used in the experiment and needs to be re-prepared.
  • mice were placed in a transparent glass cylinder (diameter: 23 cm; height: 31 cm), which was filled with 15 cm of water, and the temperature was maintained at 24 ⁇ 1 °C.
  • the FST lasts for 6 minutes, during which a high-definition camera is used to shoot.
  • Professional testing software calculates the immobility time of the mouse during the test. After the end of the test, the mouse is immediately put back into the cage, and attention should be paid to keeping warm.
  • mice were trained before the test, that is, two bottles of 1% (W/V) sucrose solution were put into each cage, and one of the bottles was replaced with pure water after 24 hours. Test after fasting for 10-24 hours after adaptation. Place 2 pre-weighed water bottles in each cage, one bottle is 1% (W/V) sucrose solution, and the other bottle is pure water. After 12 hours, take the two water bottles and weigh them again, and record the sugar water consumption and pure water consumption of each mouse. .
  • Sugar water preference index% sugar water consumption/(sugar water consumption+pure water consumption) ⁇ 100%.
  • mice After adapting to the environment, stick the tail of the mouse on the hanging rod, and keep the head of the mouse about 20-25 cm away from the ground for about 6 minutes.
  • a high-definition camera shoots a video, and uses behavioral analysis software to identify and count the immobility time of the mice. After the experiment, the mice were returned to their cages.
  • Example 20A had obvious anti-inflammatory effect.
  • NC Sodium carboxymethyl cellulose
  • provider Merrill
  • batch 69881020
  • properties white solid powder
  • quantity 200g
  • storage conditions normal temperature.
  • LPS Peripheral and central inflammation inducer
  • Experimental animal strain C57BL/6J mice; age: 6-8 weeks; gender: male; animal weight: 18-20g; number: 24.
  • Laboratory animal provider Guangdong Medical Experimental Animal Center
  • the quarantine period is 5 days, and animals with abnormal performance are removed before the experiment.
  • Animal number each cage has a cage label, indicating the number of animals, sex, strain, receiving time, group and the start time of the experiment.
  • Animal number Each animal is marked with an individual animal number on the tail.
  • the animals were randomly divided into 3 groups, and the specific grouping and treatment are shown in Table 11.
  • LPS (2mg/kg, i.p., QD) was injected on the 1st, 2nd, and 3rd day to induce peripheral and central inflammation in mice.
  • the ND drug test group was administered 2 hours before LPS injection for pre-protection (ND 30mg/kg, p.o., QD).
  • plasma and brain tissue of mice were collected for inflammatory factors and peroxidation factors and other indicators.
  • Solvent (0.5% CMC-Na solution): Take 0.5g CMC-Na white solid powder, add 100ml double distilled water, vortex until dissolved. The prepared solution should be stored in a 4-degree refrigerator and sealed. The solution can be stored for one month, but if it is found to be moldy, it is forbidden to be used in the experiment and needs to be re-prepared.
  • mice After the mice were anesthetized with 1% pentobarbital sodium, the eyeballs were removed and the blood was collected in EP tubes. After standing at 4°C for 1 hour, they were centrifuged at low temperature, and the supernatant was taken, and quickly frozen in liquid nitrogen.
  • Hippocampal tissue the mice were anesthetized with 1% pentobarbital sodium, and then the heart was perfused with cold PBS. The brain was taken out, and the tissue was taken on ice and frozen in EP tube with liquid nitrogen.
  • the frozen plasma was taken, and various indicators were tested using IL-1 ⁇ , IL-6, and IL-10 kits, and the specific operation procedures were the same as the kit instructions.
  • hippocampal supernatant was taken, and various indicators were detected using IL-1 ⁇ , IL-6, and IL-10 kits, and the specific operation procedures were the same as the instructions of each kit.
  • Example 20A As shown in Figures 24 to 29, the detection results of important indicators related to serum and hippocampal tissue inflammation in mice in each group showed that Example 20A only affected IL-1 ⁇ in serum and IL-10 in hippocampus, indicating that Example 20A (ND ) has an anti-inflammatory effect. *, P ⁇ 0.05; **, P ⁇ 0.01.
  • HEK293 cells were cultured under the condition of 5% CO 2 and the mitochondrial respiratory chain supercomplex I1III2IV1 was purified therefrom.
  • the respiratory chain super complex is chemically covalently coupled to the surface of the metal chip, small molecular compound solutions of different concentrations flow through the metal chip, and the change of the reflection coefficient of the metal surface is monitored by the Biacore 8K plus analyzer using SPR technology, and then The corresponding response curves for small molecule binding are plotted.
  • the binding strength (KD value) of the small molecule to the human mitochondrial respiratory chain supercomplex I1III2IV1 was calculated by the built-in fitting software of the Biacore 8K plus instrument.
  • Compound treatment Dissolve the small molecule shown in ddH 2 O to prepare a 10 mM stock solution.
  • 200 ⁇ L of small molecule solutions of various concentrations were taken to flow over the surface of the metal chip.
  • HEK293 cells were cultured under the condition of 5% CO 2 and mitochondria were purified therefrom, and mitochondria were sonicated to obtain SMP (submitochondrial particles). After incubating SMP with different concentrations of small molecule compounds or a control drug (Rotenone, 2 ⁇ M) for 10 minutes, 500 ⁇ M NADH was added. The absorbance of NADH was detected by the Enspire multi-mode microplate reader, the enzyme activity curve of the reduction of NADH concentration caused by SMP catalysis was drawn, and the maximum reaction rate of SMP was calculated by linear fitting. Adding 1 mM FeCN to the normal NADH catalytic system of SMP can cause a short circuit of the electronic pathway from NADH to coenzyme Q. In the presence of FeCN and Rotenone, the NADH concentration reduction curve was also determined, and the maximum reaction rate of the SMP flavin site was calculated by linear fitting.
  • Compound treatment Dissolve the small molecule shown in ddH 2 O to prepare a 10mM stock solution, and then use 10mM Tris buffer solution to dilute the small molecule gradient to 0.075, 0.1, 0.133, 0.178, 0.237, 0.316, 0.422, 0.563, 0.75, 1 ⁇ M for SMP catalysis experiments.
  • HPAEC cells human pulmonary artery endothelial cells
  • HPAEC cells human pulmonary artery endothelial cells
  • OCR oxygen consumption rate
  • HPAEC cells human pulmonary artery endothelial cells
  • Spike new coronavirus surface spike protein, 8 ⁇ g/mL
  • Example 13B pre-incubated with Example 13B (0.25, 0.5, 1 ⁇ M) for 6 hours and then incubated with Spike (8 ⁇ g/mL) for 24 hours.
  • mitochondrial staining was performed using MitoSox Red, PKMDR, and Mitotracker Green mitochondrial fluorescent probes, and fluorescent confocal photos of cells were taken by a Leica stimulated emission depletion super-resolution confocal fluorescence microscope (STED). Subsequently, ImageJ was used to measure the fluorescence intensity of cells in different groups, and the obtained data was used to calculate the standard deviation and standard error, and the T-test analysis was performed to calculate the P value to determine whether there was a significant difference.
  • mice After purchasing 6-week-old male APOE-/- mutant B6J mice and WT B6J mice, they first acclimatized in the breeding room for 2 weeks. When the mice were 8 weeks old, except the Vehicle group and the WT group, the other groups began to be given high-fat diet (Research Diets, D12492). When the mice were 9 weeks old, the Control group was given 10% cyclodextrin solution, and the rest of the administration groups Oral gavage of different concentrations of small molecule compounds was started in groups, and the frequency of gavage was once every 2 days. When the drug administration was full for 1 month, blood was collected from the orbital vein of APOE mice, and the blood collection volume was about 500mL.
  • the supernatant plasma was drawn and sent for determination of total cholesterol (CHOL), high-density lipoprotein (HDL) and Low-density lipoprotein (LDL) levels.
  • CTL total cholesterol
  • HDL high-density lipoprotein
  • LDL Low-density lipoprotein
  • the APOE mice were sacrificed and the aortic arch was dissected. After the mouse aortic arch was fixed with 4% paraformaldehyde, oil red was used to stain the plaque inside the aortic arch, and the stained aortic arch was imaged by a Zeiss stereo microscope.
  • the photos taken were analyzed with ImageJ to analyze the overall area of the longitudinal section of the arterial arch and the area of the orange-red plaque, calculate the proportion of the plaque area, and perform T-test analysis on the data to calculate the P value to determine whether there is a significant difference.
  • CRISPR Cas9 technology was used to introduce human APP mutant protein into SD rat embryos to obtain a transgenic AD rat model. Rats of this strain had amyloid deposits in the brain at the age of 2 months, and began to show certain behavioral disorders at the age of 5 months.
  • the drug was administered from the age of 7 months to the age of 13 months, and a water maze experiment was done to verify the improvement of spatial memory.
  • the process of the water maze experiment is divided into three stages: the first stage is 1 day. For the adaptation stage, there is a 15cm diameter escape platform in a large pool with a diameter of 1.8m. There are circles, triangles, The four marks of box and cross are used as hints of spatial position.
  • the rats were put into transparent water along the pool wall one by one, and the water surface was 1 cm below the escape platform. Rats will easily find the escape platform and be rescued from the water maze after climbing onto the platform, thereby learning the basic rules of the water maze.
  • the second phase of 4 days is the training phase. The setting of the pool remains the same, the water surface is raised to 1cm above the escape platform, and ink is poured in to dye the water black, making the escape platform invisible. Every day, the rats were put into the pool from four directions, southeast, northwest, and rescued after they successfully climbed onto the escape platform. If the platform is not found within 90s, the rats are manually guided to climb up the platform and rescued after standing on the platform for 15s.
  • the third stage is 1 day, which is the examination stage.
  • the escape platform was taken out, and the rat was put in once along the pool wall. Rats with strong spatial memory will repeatedly shuttle back and forth to the location of the original escape platform. Record the incubation period of the first arrival on the platform, the number of times the platform shuttles, the average distance from the platform, and the residence time in the target quadrant.
  • mice in the drug treatment group There are three groups of mice in total, 13 mice in WT group, 7 mice in model group (AD group), 11 mice in drug administration group (group B of Example 13), the latency period of reaching the platform for the first time (as shown in Figure 37a), the number of shuttles on the platform (as shown in Figure 37b), the average distance from the platform (as shown in Figure 37c), and the residence time in the target quadrant (as shown in Figure 37d), the behavioral performance of the rats in the drug treatment group was improved compared with that in the model group .
  • Figure 37e to Figure 37g show the heat maps of the movement trajectories of rats in each group in the pool.
  • mice Using CRISPR Cas9 technology, the human APP mutant protein was introduced into SD rat embryos to obtain a transgenic AD rat model. Rats of this strain had amyloid deposits in the brain at the age of 2 months, and began to show certain behavioral disorders at the age of 5 months. In order to verify the therapeutic effect, the drug was administered from the age of 7 months to the age of 15 months, and a T-maze experiment was performed to verify the improvement of working memory. The T-maze has four stages: I. Adaptation stage, food is placed in both arms, and the valves of both arms are opened. Each rat was trained twice. Enter the next stage when all the rats have eaten both sides of the food within 5 min for two times. II.
  • the forced selection training stage first close the valve on one side, and allow the rats to finish eating the food on the opposite side. Then close the valve on the opposite side, allowing the rats to finish eating the food on the other side.
  • Each animal was trained 4 times a day for 3 days. III.
  • half the valve on one side is closed first, and the rats are allowed to finish eating the food on the opposite side. Open all the valves again, and if the rat goes to the other side, it can finish the food there. If the rat went to the ipsilateral side, it was given no food and was kept off that side for 30s. This is a training session.
  • mice in the WT group There were three groups of mice in total, 3 mice in the WT group, 2 mice in the model group (AD group), and 6 mice in the administration group (Group B of Example 13). As shown in Figure 38, at 1.5 min and 3 min, there was no significant difference between the model group and the administration group, but at 10 min, the working memory of the model group was significantly weakened, while the working memory of the administration group persisted.
  • mice introduced with APP/P Example 13B were used, and the background mice were C57BL/6J.
  • the phenotype of the model mice was cognitive behavioral changes at the age of 3 months, senile plaques at the age of 5 months, and a large number of senile plaques at the age of 12 months.
  • One of the symptoms of AD patients is the inability to take care of themselves, and nesting behavior reflects the level of self-care in mice.
  • 10 rectangular pieces of paper were neatly placed in the mouse cage in order. Normal mice shredded pieces of paper to build nests, but mice with severe AD symptoms did not shred paper to build nests. Scores were made according to the degree of shredded paper and the integrity of nest building. The higher the score, the better the nesting behavior of the mice, reflecting the higher level of self-care.
  • mice introduced with APP/P Example 13B were used, and the background mice were C57BL/6J.
  • the phenotype of the model mice was cognitive behavioral changes at the age of 3 months, senile plaques at the age of 5 months, and a large number of senile plaques at the age of 12 months.
  • the process of the water maze experiment is divided into three stages: the first stage is 1 day.
  • the adaptation stage there is an escape platform with a diameter of 8 cm in a large pool with a diameter of 1.2 m. There are circles, triangles, The four marks of box and cross are used as hints of spatial position.
  • the rats were put into transparent water along the pool wall one by one, and the water surface was 1 cm below the escape platform.
  • the mice will easily find the escape platform, climb up the platform and be rescued from the water maze, thereby learning the basic rules of the water maze.
  • the second phase of 4 days is the training phase.
  • the setting of the pool remains the same, the water surface is raised to 1cm above the escape platform, and ink is poured in to dye the water black, making the escape platform invisible.
  • the mice were put into the pool from four directions, southeast, northwest, every day, and were rescued after successfully climbing the escape platform. If the platform is not found within 90s, the rats are manually guided to climb up the platform and rescued after standing on the platform for 15s.
  • the third stage is 1 day, which is the examination stage.
  • the escape platform was taken out, and the mice were still placed once along the pool wall. Rats with strong spatial memory will repeatedly shuttle back and forth to the location of the original escape platform. Record the number of platform shuttles, dwell time in the target quadrant and other indicators.
  • Results In this experiment, the drug was administered from the age of 2 months, and the water maze was used to evaluate the spatial memory 6 months after the drug was administered.
  • Four small molecules were tested, Example 23, Example 24, Example 25 and Example 26.
  • the results shown in Figures 40a and 40b show that the spatial memory ability of the model group was significantly weakened compared with the WT group, while the improvement of the spatial memory ability of Example 25 was the most obvious from the indicators such as the number of platform shuttles and the dwell time in the target quadrant.
  • mice introduced with APP/P Example 13B were used, and the background mice were C57BL/6J.
  • the phenotype of the model mice was cognitive behavioral changes at the age of 3 months, senile plaques at the age of 5 months, and a large number of senile plaques at the age of 12 months.
  • the principle of the light and dark box experiment is: mice have a natural tendency to be dark. After the mice are placed in the light box, the mice will spontaneously enter the connected dark box. But in this experiment, the dark box is equipped with an electrical stimulation mechanism, and the mice will be shocked by electric shock after entering the dark box.
  • mice no longer entered the dark box after being shocked several times, but AD mice did not remember to be shocked after entering the dark box, and would continue to enter the dark box. Therefore, after the mice were put into the light box, the latency period before the mice spontaneously entered the dark box reflected the memory strength of the mouse for the electric shock, and the later the mouse entered the dark box, the stronger the memory for the electric shock was.
  • Example 23 Example 24, Example 25 and Example 26.
  • Figure 41 compared with the WT group, the memory of electric shock in the model group was significantly weakened, while the two small molecules of Example 23 and Example 25 could effectively enhance the memory of electric shock in model mice.
  • mice which can simulate ALS (gradual freezing disease) pathogenesis. Mice of this strain generally begin to die at 90 days and reach a median of 120 days.
  • Our experiments were divided into two groups, the model group and the administration group. The model group was not administered, and the administration group was administered from the 60th day, and the small molecule Example 13B was tested at a dose of 40mpk. There were 20 mice in each group. In this experiment, the death date and body weight of each mouse were recorded, and the body weight curve and survival curve were drawn.
  • Body weight was an important index reflecting the course of ALS. As shown in Figure 42a, it can be seen that the body weight of the mice in the model group began to drop significantly at 13 weeks, while the mice in the treatment group began to drop significantly at 16 weeks. As shown in the survival curve of Figure 42b, it can be seen that a large number of mice in the model group died from 90 days, the median death was about 120 days, and all died at about 140 days. However, the administration group did not begin to die after 100 days, and a large number of patients began to die after 120 days. The median number of deaths was 135 days, and all died after 150 days. The results show that the small molecule Example 13B can effectively prolong the survival period of TDP43 A315T transgenic mice, indicating that the small molecule may effectively prolong the survival period of ALS patients.
  • mice SOD G93A transgenic mice were used.
  • the mice of this strain will simulate the pathogenesis of ALS (ALS), and will have obvious motor dysfunction as the age increases.
  • Gait analysis is a behavioral analysis method for testing motor dysfunction. Mice with motor impairments had significantly reduced stride length.
  • Our experiments were divided into two groups, the model group and the administration group. The model group was not administered, and the administration group was administered from the 60th day, and the small molecule Example 13B was tested at a dose of 40mpk. There were 10 mice in each group, at week 19, the stride length of each mouse was tested.
  • mice SOD G93A transgenic mice were used.
  • the mice of this strain will simulate the pathogenesis of ALS (ALS), and will have obvious motor dysfunction as the age increases.
  • Calculation of mean movement speed in an open field is a behavioral assay for testing motor dysfunction.
  • the average movement speed of mice with motor impairment was significantly reduced.
  • Our experiment was divided into three groups, WT group, model group and administration group.
  • the WT group was normal mice, the model group was not administered, and the administration group was administered from the 60th day to test the small molecule
  • Example 13B with a dose of 40mpk There were 10 mice in each group, and the experiment was carried out at the 14th week, and the average movement speed of each mouse in the open field was tested on time every week until the 19th week.
  • mice in good health were divided into 6 groups on average according to body weight, 10 in each group: normal control group, model group, positive drug group, different kinds of test substances (embodiment 13B, embodiment 33, embodiment 34 , both are 80mpk) group.
  • the other mice except the normal control group were given 3.0% DSS solution to drink water freely to build the model, and the model was built continuously for 8 days; the normal control group was given high-pressure sterilized filtered water to drink freely.
  • Administration began on the day of modeling, and the test substance was orally administered according to the designed dose, once a day, for 9 consecutive days. During the administration period, the body weight of the mice was monitored daily, and the shape of the feces and bleeding of the mice were observed. On the day after the last administration, after CO2 deep anesthesia, blood was collected from the heart, plasma was collected, and stored at -80°C; then the abdominal cavity was opened, the liver was removed, and stored at -80°C; finally, the entire colon tissue was removed and measured The length of the colon was photographed, and the contents of the colon were washed with normal saline after dissection along the side of the mesentery and weighed. Part of the lesion tissue was fixed with 10% formalin for HE staining for histopathological observation, and the rest of the colon tissue was frozen at -80°C.
  • the anesthesia was continued and the rats were kept in a tilted state for 15 minutes; rats in the normal control group were not stimulated by rectal catheterization. After the animals woke up, they were randomly divided into groups, and then the administration (denoted as D1) was started.
  • the blank group and the model group were given the same amount of vehicle, administered by oral gavage, once/day, for 7 consecutive days (D1-D7); daily observation Animal status, feces (loose stools, bloody stools), and body weight monitoring.
  • Leptin receptor gene (Obese Gene Receptor, OB-R) is also known as diabetes gene (Diabetes Gene, db), leptin receptor (leptin receptor, Lepr) and obesity, hypertension, diabetes, lipid Metabolic disorders are closely related. The course of the disease is strongly influenced by genetic background. Topical insulin failed to control blood glucose and elevated levels of hepatic gluconeogenesis. Using gene editing technology and embryo injection technology to construct Lepr gene mutant mice. Through blood glucose monitoring of the homozygous strain, it is found that the blood glucose level is significantly different from that of the wild control, and can be used for type II diabetes research.
  • the blood sugar of this strain of mice began to soar at the age of 8 weeks, reaching 30mmol/L, while the blood sugar of normal background mice was only about 6mmol/L.
  • the small molecules tested were Example 28, Example 29 and Example 30.
  • the subjects were all 80mpk, administered by intragastric administration once a day. There were 20 mice in each group. From the age of 8 weeks, blood glucose was monitored every week until they were sacrificed at the age of 20 weeks.
  • Example 28 and Example 29 have certain hypoglycemic effects, but Example 30 has the best hypoglycemic effect, which can reach the level of normal background mice.
  • DIO model is an obesity model caused by high-fat feeding.
  • a body composition analyzer measures body fat percentage.
  • Example 34 and Example 35 have the best weight loss effects, and can reduce the weight of obese model mice from more than 50g to the level of normal mice of about 35g.
  • Example 33, Example 36, and Example 37 also have a weight loss effect, which can be reduced to about 40g. From the point of view of body fat rate, all small molecules can effectively burn fat in mice and reduce body fat rate, and the fat burning effect of Example 34 and Example 35 is still the best, and Example 33 and Example 36 , embodiment 37 next.
  • mice 8-week-old female NOD-Scid immunodeficient mice were used as inoculated mice with Raji-Luc cells. A total of 80 mice were used for in vivo fluorescence imaging on the 9th day after inoculation. According to the fluorescence intensity, the most concentrated fluorescence intensity value in the middle was selected. 60 were used for follow-up experiments. Administration began after the first in vivo imaging, and the dosage was 100mpk once a day for intragastric administration. The small molecules tested were Example 25A, Example 25B, Example 28, Example 38, Example 39, Example 40, each Group 10. Intravital imaging was performed twice weekly after the start of dosing to observe the development of whole blood tumors in mice.
  • mice of the C57BL/6 strain were used and divided into 5 groups, including the normal control group (without injection of MPTP), the model group (using MPTP for modeling), and the treatment group of 3 small molecules ( Example 13B, Example 25A, Example 25B).
  • Drug administration was started at the same time as modeling. Modeling lasted for 5 days, 30mpk was injected intraperitoneally, administration lasted for 15 days, and 80mpk lasted for 15 days.
  • the brain was soaked and fixed in PFA. After the fixation was complete, it was transferred to sucrose dehydration. After the dehydration was complete, the brain was embedded in OCT and sectioned for TH staining.
  • the detection index is the number of TH positive cells in the substantia nigra. The more positive cells, the better the protective effect of small molecules on MPTP injury.
  • mice in each group statistical results are shown in Figure 50, T-test, **: p ⁇ 0.01, #: p ⁇ 0.05.
  • Example 13B group had a statistical difference, indicating that Example 13B had a good effect on protecting the substantia nigra neurons.
  • HEK293T cells were used to culture the new coronavirus pseudovirus that only had the ability to infect but not replicate. After the pseudoviruses in the supernatant were collected, normal HEK293T cells with ACE2 receptors on the surface were used as infection objects for pseudovirus infection. After being infected by the virus, the mitochondria in the HEK293T cells will be fragmented, which can be monitored by mitochondrial fluorescent staining (mitotracker red), indicating that the cells are destroyed by the pseudovirus, which is a model group. In the experimental group, 300nM Example 13B was used to incubate the cells for 1 hour in advance and then infected with the pseudovirus to test the protective effect of the small molecule Example 13B on virus infection.
  • TBI traumatic brain injury
  • the flow cytometry results show that the early apoptotic rate and the total apoptosis rate of the brain of the test substance embodiment 13B (80mpk) and the test substance embodiment 26 (80mpk) group rats are significantly lower than Solvent control group (P ⁇ 0.05).
  • Solvent control group P ⁇ 0.05.
  • repeated intravenous administration of the test substance Example 13B and Example 26 can significantly improve the neurological damage and apoptosis of brain cells in TBI rats with acute craniocerebral trauma, and promote the recovery of motor ability and neurological function .
  • the cell line is a pancreatic cancer cell line, which is widely used in drug sensitivity experiments.
  • Cell titer glo was used to detect cell viability, and Graphpad prism was used to draw dose-response curves. The obtained susceptibility curve is shown in the figure below.
  • Leptin receptor gene (Obese Gene Receptor, OB-R) is also known as diabetes gene (Diabetes Gene, db), leptin receptor (leptin receptor, Lepr) and obesity, hypertension, diabetes, lipid Metabolic disorders are closely related. The course of the disease is strongly influenced by genetic background. Topical insulin failed to control blood glucose and elevated levels of hepatic gluconeogenesis. Using gene editing technology and embryo injection technology to construct Lepr gene mutant mice. Through blood glucose monitoring of the homozygous strain, it is found that the blood glucose level is significantly different from that of the wild control, and can be used for type II diabetes research.
  • mice in this strain is huge, hundreds of times that of normal mice, so it can simulate patients with frequent urination.
  • the experiment is divided into three groups, model group (no administration, high urine volume morbidity mice), control group (no administration, normal mice without morbidity), and experimental group (administration embodiment 1A, 80mpk, 2 days gavage once), 2 rats in the control group, and 3 rats in the other two groups.
  • the small molecule used was Example 1A.
  • Example 1A has the function of suppressing frequent urination.
  • mice After purchasing 6-week-old male APOE-/- mutant B6J mice and WT B6J mice, they first acclimatized in the breeding room for 2 weeks. When the mice were 8 weeks old, except the Vehicle group and the WT group, the other groups began to be given high-fat diet (Research Diets, D12492). When the mice were 9 weeks old, the Control group was given 10% cyclodextrin solution, and the rest of the administration groups began to orally gavage different small molecule compounds (Example 13B, Example 33, Example 36 and Example 37) , gavage frequency once every 2 days. After 16 weeks of high-fat feeding, the APOE mice were sacrificed and the aortic arch was dissected. 100 mg/aortic vascular epithelial tissue with a fixed mass was subjected to MSD multifactor detection to determine the expression levels of various inflammatory factors, including IL10, IL-1 ⁇ , and KC/GRO.
  • MSD multifactor detection 100 mg/aortic vascular epithelial tissue
  • mice By detecting the single oral gavage administration of Example 13B, the time to fall asleep (sleep latency) and sleep duration of the ICR mice induced by a subthreshold hypnotic dose of pentobarbital sodium are affected. Whether the test substance has hypnotic effect, and compare the pharmacodynamics with the positive control drug diazepam. After the mice were adaptively fed, 50 qualified animals were selected according to body weight to enter the experiment, and they were divided into 5 groups using the Excel complete random grouping method: blank control group, positive control group (diazepam), high, medium and low doses of the test substance groups, with 10 animals in each group. Each group of animals should be given medicinal liquid and vehicle.
  • the animals of each group were intraperitoneally injected with the maximum subthreshold dose of pentobarbital sodium (at first a preliminary experiment was carried out to determine the maximum subthreshold hypnotic dose of pentobarbital sodium).
  • the time to fall asleep (latency to fall asleep) of each animal, the number of animals falling asleep within 30 minutes (the ones whose righting reflex disappeared for more than 1 minute), and the sleep duration of each mouse were recorded.
  • the positive drug group was given 1 mg/kg diazepam, which significantly prolongs the sleep of the mice; compared with the blank control group, the small molecule shown in the figure can prolong the sleep time of the mice, but there is no statistical significance In addition, the effect of the small molecule shown in the figure on prolonging the sleep time of mice was significantly weaker than that of 1 mg/kg diazepam.

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Abstract

一种菝契皂苷元类化合物在制备治疗线粒体功能异常引起的相关联疾病药物中的用途,其特征在于,所述的化合物的结构式如通式(I)所示,通过对知母皂苷元类化合物经过相关体内外模型活性测试,意外地发现很多衍生化合物具有优越的细胞保护活性,特别是对于与线粒体相关联多种适应症,尤其是多种脑神经元细胞有着出乎意料的保护活性,并且该类化合物有非常优秀的血脑通透性,对于治疗由于线粒体功能异常引起的多种疾病具有潜在的广泛用途和巨大价值,弥补了现有技术在知母皂苷元类化合物的应用的不足,具有重要科学和商业应用价值。

Description

基于菝契皂苷元结构的衍生物及其药物组合物的用途
相关申请的交叉引用
本申请主张2022年2月18日提交的申请号为202210152310.X的中国发明专利申请、2023年2月10日提交的申请号为202310093727.8的中国发明专利申请的优先权,其内容通过引用的方式并入本申请中。
技术领域
本发明涉及医药技术领域,特别涉及药物化学合成领域,具体是指一种基于菝契皂苷元类结构的衍生物及其药物组合物在制备治疗线粒体功能异常引起的相关联疾病的药物中的新用途。
背景技术
知母是我国常见的中药之一,它是百合科植物知母Anemarrhena asphodeloides Bunge的干燥块茎,具有消渴热中、除邪气、消除肢体浮肿之功效,为常用的滋阴药之一。它的提取物已经被证明具有利尿、抗糖尿病、抗血小板凝集、抗真菌、调节代谢等生物活性,而且也表现出对环腺苷酸磷酸二酯酶的抑制作用。提取物主要的化学成分有甾体皂苷、双苯吡喃酮类、多糖类和木质素类等。其中甾体皂苷又包括知母皂苷A-I、A-II、A-III、A-IV、B-I、B-II和B-III,以及马尔考皂苷元3-O-β-D-吡喃葡糖基(1→2)-β-D-吡喃半乳糖苷B、去半乳糖替告皂苷、F-芝脱皂苷和异菝契皂苷等。此外,还含有知母多糖A/B/C/D、顺-扁柏树脂酚、单甲基-顺-扁柏树脂酚、氧化-顺-扁柏树脂酚、2,6,4’-三羟基-4-甲氧基二苯甲酮、对-羟苯基巴豆油酸、二十五烷酸乙烯脂、β-谷甾醇、芒果苷、烟酸、烟酰胺及泛酸等。
线粒体功能和行为是人类生理学的核心,线粒体执行多种且相互关联的功能,产生ATP和许多生物合成同时也有助于细胞应激反应,如自噬和细胞凋亡。线粒体形成一个动态的、相互联系的网络,并紧密结合在一起与其他细胞隔室一起。此外,线粒体功能超出细胞边界,并通过调节细胞间的通讯影响生物体的生理学和组织。线粒体呼吸链主要由线粒体呼吸链酶组成,线粒体呼吸链酶复合物缺陷是导致线粒体病的重要原因(约有30%-40%的线粒体疾病是由于线粒体呼吸链酶缺陷造成)。人源呼吸链超超级复合物的结构与功能,氧化磷酸化由位于线粒体内膜上的五种呼吸链蛋白复合物分步完成,这五种蛋白复合物分别为复合物I(NADH脱氢酶)、复合物II(琥珀酸脱氢酶)、复合物III(细胞色素c还原酶)、复合物IV(细胞色素c氧化酶)、和复合物V(ATP合酶)。
因此,线粒体功能障碍成为一个关键因素并不令人惊讶包括神经退行性疾病和代谢紊乱在内的多种疾病的发病因素。文献也介绍了线粒体生物学的进展并讨论它们与人类疾病的相关性(doi:10.1016/j.cell.2012.02.035.)。目前已经有多种小分子药物都以呼吸链复合物为靶点,并且作用于多种适应症,为疾病治愈提供了多种选择。各类适应症目前已有的靶向呼吸链复合物的药物数量,包括:肿瘤癌症、心血管疾病、内分泌疾病、免疫疾病、冠状病毒感染、炎症、代谢类疾病、神经退行性疾病、肠道疾病、自闭症、精神分裂症、老年痴呆症、帕金森病、癫痫、中风、、慢性疲劳综合症等。
小分子作用在线粒体可以使复合物维持在活性状态,并且别构调控复合物的活性和电子传递效率,同时有助于复合物整体的结构稳定,从而减少电子泄露导致的线粒体ROS(超氧自由基)产生。基于对复合物活性的调节,可以达到对细胞乃至机体内能量代谢通路的整体调控,以及对基因组的表观遗传修饰进行影响。
脑卒中俗称中风,包括缺血性脑卒中(脑梗死)和出血性脑卒中(脑实质出血、脑室出 血、蛛网膜下腔出血)。根据世界卫生组织定义,脑卒中多种原因导致脑血管受损,局灶性(或整体)脑组织损害,引起临床症状超过24小时或致死。具有发病率、致残率、复发率和死亡率高的特点。脑卒中是中国居民第一位死亡原因。
肌萎缩侧索硬化症也叫运动神经元病,它是上运动神经元和下运动神经元及其支配的躯干、四肢和头面部肌肉的,一种慢性、进行性变性疾病。常表现为上、下运动神经元合并受损所致的进行性加重的肌无力、肌萎缩、肌束颤动等。临床表现为进行性加重的骨骼肌无力、肌萎缩、肌束颤动、延髓麻痹和锥体束征,早期患者症状轻微,易与其他疾病混淆,患者可能只是感到一些无力、肉跳、容易疲劳等一些症状,渐渐进展为全身肌肉萎缩和吞咽困难,最后产生呼吸衰竭。产生运动神经元损害的原因,目前主要理论有:1,神经毒性物质累计,谷氨酸堆积在神经细胞之间,久而久之,造成神经细胞的损伤;2,自由基使神经细胞膜受损;3,神经生长因子缺乏,使神经细胞无法持续生长、发育。目前国际承认、且唯一通过美国食品药物监督局批准治疗肌萎缩侧索硬化的药物为力如太(Rilutek),并且一定要尽早使用。同时,国际上也正在尝试以神经营养因子、抗氧化剂如维生素E、维生素C以及肌酸、CoQ10等与力如太联合应用,以对肌萎缩侧索硬化进行保护性治疗,但还有待于临床实试验的证实。
神经退行性疾病分为急性神经退行性疾病以及慢性神经退行性疾病。急性神经退行性疾病主要包括脑卒中、脑损伤(BI)及癫痫等;慢性神经退行性疾病主要包括阿尔茨海默病(AD)、帕金森病(PD)、亨廷顿病(HD)、肌萎缩性侧索硬化(ALS)、不同类型脊髓小脑共济失调(SCA)及Pick病等。神经退行性疾病的成因主要有以下四方面:1,氧化应激。氧化应激是由于自由基过度产生和(或)得不到及时清除、体内氧化与抗氧化作用失衡所致,机体的细胞和组织损伤。自由基是具有不成对电子的原子或基团,包括羟自由基、超氧阴离子、一氧化氮等。近年来在神经退行性疾病如AD、PD、ALS中均发现有神经组织的氧化损伤;2,线粒体功能障碍。AD患者脑内存在mtDNA缺陷和氧化磷酸化异常。聚合酶链式反应(PCR)及印迹杂交检测发现,散发型AD患者脑组织出现mtDNA断裂、碱基缺失及错译突变。电镜观察证实线粒体数目增加,结构异常,出现层状体和晶状包涵体。另外,AD患者神经元线粒体功能发生障碍后,将导致神经元能量供给不足同时释放大量ROS,诱发氧化应激损伤,钙调节失衡,最终触发神经元凋亡;3,兴奋性毒素。细胞间隙中谷氨酸浓度过高时会对神经元产生毒素,导致神经元的退化、衰老及死亡。谷氨酸的这种兴奋性毒性作用与多种神经退行性疾病的发生、发展都有密切联系,是导致神经退行性疾病中神经细胞死亡的重要机制之一;4,免疫炎症。许多证据都表明炎症在AD发病中占有重要的地位。固有免疫系统是在种系发育和进化过程中形成的天然免疫防御功能。与机体另一种特异性免疫应答相比,它能对各种有害物质进行迅速的反应,以保护机体。固有免疫系统本身的激活是一把双刃剑。有害物质(如聚集形式的Aβ)长期和不可控制的刺激,启动固有免疫系统,就会产生对大脑的损害作用。
新型冠状病毒肺炎(Corona Virus Disease 2019,COVID-19),简称“新冠肺炎”,世界卫生组织命名为“2019冠状病毒病”,是指2019新型冠状病毒感染导致的急性呼吸道传染病。2020年3月11日,世卫组织评估后认为当前新冠肺炎疫情可被称为全球大流行。新冠病毒感染的肺炎患者的临床表现为:以发热、乏力、干咳为主要表现,会出现缺氧低氧状态,约半数患者多在一周后出现呼吸困难,严重者快速进展为急性呼吸道窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍。目前新冠的预防方法主要是疫苗和隔离,在治疗药品方面包括Paxlovid、阿兹夫定片、莫诺拉韦胶囊、散寒化湿颗粒,清肺排毒汤等。
寻找高效、高选择性、低毒、脑通透性好并且急需的新型药物仍然极具挑战性,预防,降低和治疗新冠病毒感染,降低死亡率和消除后遗症,相当迫切。降低脑内自由基水平被认为是一种治疗手段,知母皂苷元类化合物已被证明具有降低自由基水平,保护线粒体使其更好有效地利用氧气的作用。
因此,用于制备治疗线粒体呼吸链异常引起的疾病方面的化合物开发并合理使用,是非 常有实用价值的。
发明内容
本发明的目的是为了克服上述现有技术中的缺陷,为了实现上述目的,本发明第一方面提供了一种基于菝契皂苷元结构的衍生物在制备治疗线粒体功能异常引起的相关联疾病的药物中的用途,其主要特点是,所述的衍生物的结构式如通式I所示,
所述的通式I所示的衍生物由以下片段A和片段B连接而成,
其中,Z为NR1R2;R1和R2各自独立地为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、氨基、硝基、氰基、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基、苯基、苄基、C3-C14杂环基、C3-C14杂芳基,杂原子选自N、O、S的一个或多个;或者R1和R2一起形成三至八元环,所述的三至八元环具有一个或多个选自C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3-C14杂芳基、卤素、羟基、氨基、烷氧基、-CF3、-SF5或杂原子为硫、氧、NH或NRa的三至八元杂环的取代基,杂原子选自N、O、S的一个或多个;
X为C(O)或S(O)2
Y为C(Rd)(Re)、C(O)或S(O)2,Rd、Re各自地独立为氢或具有至少一个取代基的C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3-C14杂芳基,其中的取代基选自卤素、羟基、氨基、硝基、氰基、醛基、羧基、烷氧基、-CF3或-SF5,杂原子选自N、O、S的一个或多个,或者,Rd和Re一起形成三至八元环,所述的三至八元环具有一个或多个选自C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3-C14杂芳基、卤素、羟基、氨基、烷氧基、-CF3、-SF5或杂原子为硫、氧、NH或NRa的三至八元杂环的取代基,杂原子选自N、O、S的一个或多个;
X2为O、S或NH;
Ra独立地为氢或具有至少一个取代基的C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3- C14杂芳基,其中的取代基选自卤素、羟基、氨基、硝基、氰基、烷氧基、-CF3或-SF5,杂原子选自N、O、S的一个或多个;
n为0至10的整数且n不为0,m为1,或者,n为0,m为1,或者,n为0至10的整数且n不为0,m为0;
R3、R4a、R4b、R5a、R5b各自独立地为氢或选自卤素、取代的烷基、羟基、氨基;
表示单键或者双键;
各“*”独立地表示消旋、S或R构型。
优选地,所述的衍生物的结构式如通式(II)所示,
优选地,所述的衍生物的结构式如通式III所示,
优选地,所述的衍生物的结构式如通式IV所示,
优选地,所述的衍生物的结构式如通式V所示,
其中,R6为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基、苯基、苄基,吡啶基,-CO烷基,-CO芳香基、-SO2烷基、-SO2芳香基、-CO2烷基、C2-C4(CO)链烯基、-CO2芳香基、-SO3H;
L为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基;
n为0至10的整数;
n2为0,1,2,或3;
m,m’独立地为1至4的整数;
W1为C或NH;
V1为C或NH;
M为C、S、O或NH;
优选地,所述的衍生物的结构式如通式VI所示,
Y1为C(Rd)(Re)、C(O)或S(O)2、Rd、Re独立地为氢或具有至少一个取代基的C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3-C14杂芳基,其中的取代基选自卤素、羟基、氨基、硝基、氰基、醛基、羧基、烷氧基、-CF3或-SF5、杂原子选自N、O、S的一个或多个,或者,Rd和Re一起形成三至八元环,所述的三至八元环具有一个或多个选自C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3-C14杂芳基、卤素、羟基、氨基、烷氧基、-CF3、-SF5或杂原子为硫、氧、NH或NRa的三至八元杂环的取代基、杂原子选自N、O、S的一个或多个;
L为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基;
n为0至10的整数;
n2为0,1,2,或3;
n3为1至10的整数,
m为0至10的整数。
优选地,所述的衍生物的结构式如通式VII所示,
其中,R6、R7独立地为氢、取代或未取代的C1-C10烷基、C1-C10烷基的取代基选自卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基、苯基、苄基、吡啶基、-CO烷基、-CO芳香基、-SO2烷基、-SO2芳香基、-CO2烷基、C2-C4(CO)链烯基、-CO2芳香基、-SO3H;
L为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基;
W2为C或NH;
V2为C、O、S或NH;
n为0至10的整数;
n1为1到10的整数;
n2为0,1,2,或3。
优选地,所述的衍生物的结构式如通式VIII所示,

Z1为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基、苯基、苄基、吡啶基、-CO烷基、-CO芳香基、-SO2烷基、-SO2芳香基、-CO2烷基、C2-C4(CO)链烯基、-CO2芳香基,-SO3H;
W3为C、S、O或NH;
n为0到10的整数;
n4,n5,n6,n7为1至4的整数。
在另一优选例中,所述的衍生物的结构式中片段B为如下结构:
优选地,所述的衍生物为以下化合物、以下化合物的非对映异构体混合物或以下化合物 的对映异构体中的一种,



































优选地,所述的衍生物包括其上任何一个或多个氢原子被其稳定同位素氘取代而产生的相应氘代化合物。
本发明的另一方面,提供一种药物组合物,所述药物组合物包含:上述的通式I化合物、其药学上可接受的盐、立体异构体、互变异构体、前药或其药学上可接受的载体。
优选地,还包括附加治疗剂,所述的附加治疗剂包括抗抑郁药、抗狂躁药、帕金森病治疗药、阿尔兹默病治疗药或它们的组合。
优选地,所述药学上可接受的盐选自下组:盐酸盐、氢溴酸盐、硫酸盐、磷酸盐、甲磺酸盐、三氟甲磺酸盐、苯磺酸盐、对甲苯磺酸盐(甲苯磺酸盐)、1-萘磺酸盐、2-萘磺酸盐、乙酸盐、三氟乙酸盐、苹果酸盐、酒石酸盐、柠檬酸盐、乳酸盐、草酸盐、琥珀酸盐、富马酸 盐、马来酸盐、苯甲酸盐、水杨酸盐、苯乙酸盐、扁桃酸盐。
优选地,所述的附加治疗剂为吗氯贝胺、托洛沙酮、氟西汀、帕罗西汀、西酞普兰、舍曲林、文拉法辛、曲米帕明、曲唑酮、丙咪嗪、地昔帕明、氯米帕明、阿米替林、去甲替林、多塞平、马普替林、洛沙平、阿莫沙平、米氮平、丁螺环酮、氯美扎酮、坦度螺酮、碳酸锂、他克林、石杉碱甲、加兰他敏、多奈哌齐、力帆斯的明、美金刚、普拉克索、他利克索、罗匹罗尼,或它们的组合。
本发明还提供了所述的药物组合物在制备防护、处理、治疗或减轻患者疾病、病症或病状的药物中的应用。
优选地,所述的线粒体异常引起的相关联疾病、病症或病状具体为呼吸链异常引起的相关联疾病、病症或病状,包括:代谢类疾病、肿瘤、炎症、中枢神经系统性疾病四个大类。
代谢类疾病包括:高血糖、高血脂、高胆固醇、高低密度脂蛋白、低高密度脂蛋白,血管生成性病症、非酒精性脂肪肝性肝、脑血管意外、心肌梗死、动脉粥样硬化、冠心病、抗衰老、尿急尿频、I型糖尿病、慢性阻塞性肺病等。
肿瘤包括:前列腺增生、韦格纳肉芽肿、肺结节病、白血病、淋巴瘤、胰腺癌、神经肿瘤等。
炎症包括:外周神经炎、化疗诱导的外周神经炎、自体免疫疾病、与器官移植相关联的病状、流感病毒、冠状病毒(感染的预防、治疗及后遗症消除)、急性呼吸窘迫综合征、炎性肠病、克罗恩氏病、溃疡性结肠炎、银屑病、视网膜脱离、色素性视网膜炎、黄斑变性、胰腺炎、特应性皮炎、类风湿性关节炎、脊椎关节炎、痛风、系统性红斑狼疮、干燥综合症、全省性硬皮病、抗磷脂综合征、血管炎、骨关节炎、自身免疫性肝炎、自身免疫性肝胆疾病、原发性硬发性胆管炎、肾炎、乳糜泻、自身免疫ITP、移植排斥、实体器官的缺血再灌注损伤、败血症、牙周炎、全身性炎症反应综合症、心肌炎、变应性疾病、哮喘、白细胞介素-I转化酶相关的发热综合征、白塞氏病等。
中枢神经系统性疾病包括:皮克病、脊髓损伤修复、抑郁症、焦虑症、帕金森病、阿尔兹海默病、睡眠障碍、缺血性脑卒中、出血性脑卒中、肌肉萎缩性侧索硬化症、外伤性脑损伤、脑萎缩、亨廷顿病、精神分裂症、躁狂症、毒瘾戒断、多发性硬化症、改善睡眠、肌无力等。
采用本发明基于菝契皂苷元类的化合物、药物组合物及其应用,通过对知母皂苷元类化合物经过相关体内外模型活性测试,意外地发现很多衍生化合物具有优越的细胞保护活性,特别是对于多种脑神经元细胞有着出乎意料的保护活性,并且该类化合物有非常优秀的血脑通透性,对于治疗多种线粒体功能异常引起的疾病具有潜在的广泛用途和巨大价值,弥补了现有技术在知母皂苷元类化合物的应用的不足,具有重要科学和商业应用价值。
附图说明
图1为不同小分子化合物作用下神经元细胞死亡情况。
图2为小分子化合物对过氧化氢(H2O2)诱导人SHSY5Y神经肿瘤细胞氧化损伤情况。
图3为神经元死细胞比例。
图4为各实验组对斑马鱼炎症的影响表型图。
图5为各实验组对斑马鱼炎症的影响(中性粒细胞个数)。
图6为各实验组对炎症斑马鱼的抗炎作用。
图7为术中小鼠脑血流变化。
图8为小鼠术前术后体重变化。
图9为小鼠前肢抓力变化。
图10为小鼠神经功能缺损评分。
图11为小鼠脑梗死体积。
图12为小鼠脑水肿体积。
图13为小鼠FST不动时间。
图14为小鼠SPT中糖水偏好率。
图15为小鼠TST不动时间。
图16为小鼠血清中ROS水平检测。
图17为小鼠血清中H2O2浓度检测。
图18为小鼠血清中NO浓度检测。
图19为小鼠血清中脂质过氧化水平检测。
图20为小鼠海马中ROS水平检测。
图21为小鼠海马中H2O2浓度检测。
图22为小鼠海马中NO浓度检测。
图23为小鼠海马中脂质过氧化水平检测。
图24为小鼠血清中IL-1β浓度检测。
图25为小鼠血清中IL-6浓度检测。
图26为小鼠血清中IL-10浓度检测。
图27为小鼠海马中IL-1β浓度检测。
图28为小鼠海马中IL-6浓度检测。
图29为小鼠血清中IL-10浓度检测。
图30a至32b分别为各小分子与呼吸链复合物I的结合强度。
图33为各小分子对SMP的活性结果图。
图34为各小分子对细胞耗氧的结果示意图。
图35a至35b分别为小分子对线粒体ROS和线粒体跨膜电势差的结果图。
图36a至36d分别为小分子对APOE小鼠动脉粥样硬化的结果图。
图37a至37g为小分子对AD大鼠水迷宫结果图。
图38为小分子对AD大鼠T迷宫结果图。
图39a和39b为小分子对AD小鼠筑巢行为的结果图。
图40a和40b为小分子对AD小鼠水迷宫结果图。
图41为小分子对AD小鼠明暗箱结果图。
图42a和42b为小分子对TDP43A315T小鼠生存期的结果图。
图43为小分子对SODG93A小鼠步态分析结果图。
图44为小分子对SODG93A小鼠旷场结果图。
图45a和45b为小分子对DSS小鼠肠炎模型的结果图。
图46a和46b为小分子对TNBS大鼠肠炎模型的结果图。
图47为小分子对DB小鼠血糖的结果图。
图48a至48d为DIO小鼠体重、体脂率的结果图。
图49为小分子对血液瘤的体内杀伤效果图。
图50为小分子对黑质神经元保护作用的结果图。
图51为小分子对病毒侵染的保护作用的结果图。
图52为小分子对大鼠TBI急性颅脑创伤模型的结果图。
图53为小分子对胰腺癌细胞的体外杀伤效果的结果图。
图54为小分子对DB动物过量排尿的结果图。
图55a至55c为小分子对高脂饲喂的APOE突变小鼠的心血管炎症的结果图。
图56a和56b为小分子对阈下催眠剂量戊巴比妥钠影响小鼠睡眠的药效实验结果图。
具体实施方式
为了能够更清楚地理解本发明的技术内容,下面对本发明的具体实施方法作进一步说明。
本发明中的术语“烷基”,是指一价饱和脂族烃基,具有1至10个碳原子,包括直链和支链烃基,如甲基(CH3-)、乙基(CH3CH2-)、正丙基(CH3CH2CH2-)、异丙基((CH3)2CH-)、正丁基(CH3CH2CH2CH2-)、异丁基((CH3)2CHCH2-)、仲丁基((CH3)(CH3CH2)CH-)、叔丁基((CH3)3C-)、正戊基(CH3CH2CH2CH2CH2-)、新戊基((CH3)3CCH2-)。
在本发明中,术语“烷基”包括取代或未取代的烷基。
在本发明中,术语“取代或未取代的”指所述基团可以是未取代的,或者所述基团中的H被一个或多个(较佳地1~6个,更佳地1~3个)取代基所取代。
在本发明中,所述的“取代的”指所述基团具有一个或多个(较佳地1~6个,更佳地1~3个)选自下组的取代基:卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基、苯基、苄基、C2-C8杂环基、C2-C8杂芳基,杂原子选自N、O及S中的一个或多个。
在本发明中,术语“环烷基”表示取代或未取代的C3-C12环烷基。
在本发明中,术语“烷氧基”指-O-烷基,其中所述烷基可以是饱和或不饱和的、可以是支链、直链的、或环状的。优选地,烷氧基具有1~10个碳原子,较佳地1~6个碳原子。代表性的例子包括(但并不限于):甲氧基、乙氧基、丙氧基。
在本发明中,术语“芳基”是指6至20个(较佳6~14个)碳原子的单价芳香族碳环基团,它具有单环(如苯基)或稠环(如萘基或蒽基),如果连接点在芳香碳原上,稠环可能是非芳香性的(如2-苯并噁唑酮,2H-1,4-苯并噁嗪-3(4H)-酮-7-基等)。优选的芳基包括苯基和萘基。该术语包括取代或未取代的形式,其中取代基的定义如上。
在本发明中,术语“烯基”是指具有2至10(如2至6或2至4)个碳原子的烯基,且具有至少1(如1至2)个不饱和烯键(>C=C<)。这类基团的例如有乙烯基、烯丙基、丁-3-烯基。
在本发明中,术语“环烷基”是指具有3至10个碳原子的、具有单环或多环(包括稠合体系,桥环体系和螺环体系)的环状烷基。在稠环体系中,一个或多个环可以是环烷基、杂环的、芳基或杂芳基,只要连接位点是通过环烷基的环。合适的环烷基的例子包括:例如,金刚烷基、环丙基、环丁基、环戊基和环辛基。
在本发明中,术语“卤代”或“卤素”是指氟、氯、溴和碘。
在本发明中,术语“杂芳基”是指环内具有1至10个碳原子和1至4个选自氧、氮和硫的杂原子的芳香基团,这样的杂芳基可以是单环的(如吡啶基或呋喃基)或稠环(如吲嗪基(indolizinyl)或苯并噻吩基),其中,所述稠环可以是非芳香性的和/或含有一个杂原子,只要连接点是通过芳香性杂芳基的原子。在一实施例中,杂芳基的环原子氮和/或硫任选地被氧化为N-氧化物(N-O),亚磺酰基或磺酰基。优选地杂芳基包括吡啶基、吡咯基、吲哚基、噻吩基和呋喃基。该术语包括取代或未取代的杂芳基。
在本发明中,术语“取代的杂芳基”是指被1至5个、优选1至3个、更优选1至2个的取代基所取代的杂芳基,所述取代基选自与取代的芳基所定义的相同取代基。
在本发明中,术语“杂环”或“杂环的”或“杂环烷基”或“杂环基”是指饱和的、部分饱和的或不饱和的基团(但不是芳香性的),具有单环或稠环(包括桥环体系和螺环体系),环内具有1至10个碳原子和1至4个(如3个)选自氮、硫或氧的杂原子,在稠环体系中,一个或多个环可以是环烷基、芳基或杂芳基,只要连接点通过非芳香性环。在一实施例中,杂环基团的氮原子和/或硫原子任选地被氧化,以提供N-氧化物、亚磺酰基和磺酰基部分。
在本发明中,术语“取代的杂环的”或“取代的杂环烷基”或“取代的杂环基”是指被1到5(如1至3)个取代基所取代的杂环基团,所述取代基与取代的环烷基所定义的取代基相同。
在本发明中,术语“立体异构体”是指一个或多个立体中心的手性不同的化合物。立体异 构体包括对映异构体和非对映异构体。
在本发明中,术语“互变异构体”是指质子位置不同的化合物的替代形式,如烯醇-酮和亚胺-烯胺互变异构体,或杂芳基的互变异构形式,所述杂芳基包含与环的-NH-部分和环的N-部分连接的环原子,如吡唑、咪唑、苯并咪唑、三唑和四唑。
本发明提供了一种药物组合物,它包含安全有效量范围内的活性成分,以及药学上可接受的载体。
本发明所述的“活性成分”是指本发明所述的通式(I)化合物或其药学上可以接受的盐、其立体异构体或其互变异构体、或其前药。
本发明所述的“活性成分”和药物组合物可用作线粒体保护剂。在另一优选例中,用于制备预防和/或治疗神经退行性疾病的药物。在另一优选例中,用于制备预防和/或治疗与线粒体相关的代谢类疾病的药物。
“安全有效量”指的是:活性成分的量足以明显改善病情,而不至于产生严重的副作用。通常,药物组合物含有1~2000mg活性成分/剂,更佳地,含有10~200mg活性成分/剂。较佳地,所述的“一剂”为一个药片。
“药学上可接受的载体”指的是:一种或多种相容性固体或液体填料或凝胶物质,它们适合于人使用,而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的活性成分以及它们之间相互掺和,而不明显降低活性成分的药效。
本发明优选实施例的化合物可以作为单独活性药剂给药,也可以与一个或多个其它用于治疗癌症的试剂组合使用。
通常,优选实施例的化合物将以治疗有效量、通过具有类似作用的药剂的任意一种可接受的模式施用。优选实施例的化合物(即活性成分)的实际用量根据多个因素确定,如待治疗疾病的严重程度、患者的年龄和相对健康程度、被使用化合物的效力、施用的路径和形式,以及其他因素。该药物可一天施用多次,优选地,每天一次或两次。所有这些因素都在主治医生的考虑范围内。
优选实施例的目的,治疗有效剂量通常可以是对患者一次性施用或分次施用的每日总剂量,例如,每日约0.001至约1000毫克/公斤体重,优选地,每日约1.0至约30毫克/千克体重。单位剂量组合物(Dosage unit composition)可包含其剂量因数以形成每日剂量。剂型的选择取决于各种因素,例如给药模式和药物物质的生物利用度。通常,优选实施例的化合物可作为药物组合物通过以适宜所治疗病况的任何途径施用。适宜的途径包括但是不局限于口腔、胃肠外(包括皮下、肌肉、静脉内、动脉内、皮内)、阴道、腹膜内、肺内和鼻内。应当理解,优选的途径可以因病人的病况变化。优选的给药方式为口服,可根据苦的程度调节方便的日剂量。可以将其与药学上可接受的载体或赋型剂配制成片剂、丸剂、胶囊、半固体、粉剂、缓释制剂、溶液、悬浮液、酏剂、气雾剂或任何其他适当的组合物等。当所述化合物配制成胃肠外时,其可以与药学上可接受的胃肠外载体配制。另一种优选的施用优选实施例化合物的方式为吸入。这是一种将治疗剂直接运送给呼吸道的有效方法(参见,如美国专利号5,607,915)。
本发明可以以任意方便的制剂形式施用化合物,本发明所称的“制剂”是指含有本发明通式I化合物的有利于给药(drug delivery)的剂型,如:但不仅限于,水溶液注射剂、粉针剂、丸剂、散剂、片剂、贴剂、栓剂、乳剂、霜剂、凝胶剂、颗粒剂、胶囊剂、气雾剂、喷雾剂、粉雾剂、缓释剂和控释剂等。这些药用辅料既可以是各种制剂中常规使用的,如:但不仅限于,等渗剂、缓冲液、矫味剂、赋型剂、填充剂、粘合剂、崩解剂和润滑剂等;也可以是为了与所述物质相适应而选择使用的,如:乳化剂、增溶剂、抑菌剂、止痛剂和抗氧剂等,这类辅料能有效提高组合物所含化合物的稳定性和溶解性或改变化合物的释放速率和吸收速率等,从而改善本发明化合物在生物体内的代谢,进而增强给药效果。此外,还可以为实现特定的给药目的或方式,如:缓释给药、控释给药和脉冲给药等,而使用的辅料,如:但不仅限于, 明胶、白蛋白、壳聚糖、聚醚和聚酯类高分子材料,如:但不仅限于,聚乙二醇、聚氨酯、聚碳酸酯及其共聚物等。所称的“有利于”的主要表现有但不仅限于提高治疗效果、提高生物利用度、降低毒副作用和提高患者顺应性等。
合适的药学上可接受的载体或赋形剂包括:如处理剂和药物运送改性剂和促进剂,诸如磷酸钙、硬脂酸镁、滑石、单糖、二糖、淀粉、明胶、纤维素、甲基纤维素、羧甲基纤维素钠、葡萄糖、羟丙基-β-环糊精、磺丁基-β-环糊精钠、聚乙烯吡咯烷酮、低熔点蜡、离子交换树脂等,及其任意两种或多种的组合。液体和半固体的赋形剂可以选自甘油、丙二醇、水、乙醇和各种油,包括石油、动物油、植物油或合成来源,如花生油、豆油、矿物油、芝麻油等。优选的液体载体,特别是用于可注射溶液的载体,包括水、盐水、葡萄糖水性溶液和乙二醇。其它适宜的药学上可接受的赋形剂在《雷明顿药物科学》(Remington’s Pharmaceutical Sciences),Mack Pub.Co.,新泽西(1991)有描述,通过引用纳入本文。
在本发明中,术语“药学上可接受的盐”是指通式I化合物的非毒性酸或碱土金属盐。这些盐可在最终分离和纯化通式I化合物时原位制得、或分别将合适的有机或无机酸或碱与碱性或酸性官能团反应制得。代表性的盐包括,但不限于:乙酸盐、己二酸盐、藻酸盐、柠檬酸盐、天冬氨酸盐、苯甲酸盐、苯磺酸盐、硫酸氢盐、丁酸盐、樟脑酸盐、樟脑磺酸盐、二葡糖酸盐、环戊烷丙酸盐、十二烷基硫酸盐、乙磺酸盐、葡萄糖庚酸盐、甘油磷酸盐、半硫酸盐、庚酸盐、己酸盐、富马酸盐、盐酸盐、氢溴酸盐、氢碘酸盐、2-羟基乙磺酸盐、乳酸盐、马来酸盐、甲磺酸盐、烟酸盐、2-萘基磺酸盐、草酸盐、双羟萘酸盐、果胶酸盐、硫氰酸盐、3-苯基丙酸盐、苦味酸盐、新戊酸盐、丙酸盐、琥珀酸盐、硫酸盐、酒石酸盐、硫氰酸盐、水杨酸盐、对甲苯磺酸盐和十一烷酸盐。此外,含氮的碱性基团可被如下试剂季铵盐化:烷基卤化物,如甲基、乙基、丙基、丁基的氯化物、溴化物和碘化物;二烷基硫酸盐,如二甲基、二乙基、二丁基和二戊基硫酸酯;长链卤化物如癸基、月桂基、肉豆蔻基和硬脂基的氯化物、溴化物和碘化物;芳烷基卤化物如苄基和苯乙基溴化物等。由此得到水溶性或油溶性或可分散产品。可被用于形成药学上可接受的酸加成盐的酸的例子包括如盐酸、硫酸、磷酸的无机酸,和如草酸、马来酸、甲磺酸、琥珀酸、柠檬酸的有机酸。碱加成盐可在最终分离和纯化通式I的化合物时原位制得、或使羧酸部分分别与合适的碱(如药学上可接受的金属阳离子的氢氧化物,碳酸盐或碳酸氢盐)或氨、或有机伯、仲或叔胺反应制得。药学上可接受的盐包括,但不限于,基于碱金属和碱土金属的阳离子,如钠、锂、钾、钙、镁、铝的盐等,以及无毒的铵、季铵和胺阳离子,包括,但不限于:铵、四甲基铵、四乙基铵、甲胺、二甲胺、三甲胺、三乙胺、乙胺等。其它代表性的用于形成碱加成盐的有机胺包括二乙胺、乙二胺、乙醇胺、二乙醇胺、哌嗪等。
在本发明中,术语“药学上可接受的前药”是指那些优选实施例的化合物的前药,在体内迅速转化为上述通式所示的母体化合物的化合物,例如在血液中水解。在“T.Higuchi和V.Stella,作为新型运送系统的前药(Pro-drugs as Novel Delivery Systems),A.C.S.15 Symposium Series的14卷”和“Edward B.Roche编,药物设计中的生物可逆载体(Bioreversible Carriers in Drug Design),美国药学协会和Pergamon出版社,1987年”中提供了完整的讨论,这两者都引入本文作为参考。
本发明提供了通式(I)化合物的制备方法。以知母皂苷元举例,关键中间体的制备方法如下:
α构型:
方法一:
方法二:
β构型:
方法一:
方法二:
混合构型:
通式(I)化合物的制备如下:以知母皂苷元母核α构型为例(其他构型或者其他母核的某种构型,制备方法均与提供的方法一致,合成路线如下:
方案1:
方案2:
方案3:
各式中,R1、R2、Y、n的定义如上所示。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
下面的缩写具有如下所示的意义:
DBU是指1,8-二氮杂双环[5.4.0]十一碳-7-烯;DIBAL表示二异丁基氢化铝;DIAD指偶氮二甲酸二异丙脂;DIEA指二异丙基乙胺;DMAP是指N,N-二甲基氨基吡啶;DME指1,2-二甲氧基乙烷;DMF指N,N-二甲基甲酰胺;DMPE是指1,2-双(二甲基膦基)乙烷;DMSO表示二甲亚砜;DPPB指1,4-双(二苯基膦基)丁烷;DPPE指1,2-双(二苯基膦基)乙烷;DPPF指1,1'-双(二苯基膦基)二茂铁;DPPM指1,1'-双(二苯基膦基)甲烷EDC表示1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐;HATU表示2-(7-氮杂-1H-苯并三唑-1-基)-1,1,3,3-四甲基脲六氟磷酸盐;HMPA表示六甲基磷酰胺;IPA是指异丙醇;LDA是指二异丙基氨基锂;LHMDS是指二(三甲基硅基)氨基锂;LAH表示氢化铝锂;PyBOP是指苯并三唑-1-基-氧基三吡咯烷基磷苯并三唑六氟磷酸盐;TDA-I是指三(2-(2-甲氧基乙氧基)乙基)胺;DCM指二氯甲烷;TEA是指三乙胺,TFA是指三氟乙酸;THF是指四氢呋喃;NCS指N-氯琥珀酰亚胺;NMM是指N-甲基吗啉;NMP是指N-甲基吡咯烷酮;PPh3指三苯基膦,T3P指1-丙基磷酸环酐,PMA指磷钼酸,PE指石油醚,EA指乙酸乙酯;RBF是指圆底烧瓶;r.t是指室温。
除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1
制备过程具体如下:
第一步:中间体1
在500mL圆底烧瓶中加入10克原料A,用DCM(100mL)溶解,0℃下加入戴斯马丁(12.8g),15min后升至r.t.搅拌反应1h,TLC跟踪反应结束(PMA显色)。反应毕,抽滤除去滤渣,滤液旋干后用DCM复溶,水洗2次,干燥,硅胶柱层析得9g白色固体产品(中间体1)。
第二步:中间体2
在100mL圆底烧瓶中加入5克中间体1和1.7克NH2OH·HCl,用干燥的50mL Pyridine溶解,70℃下搅拌反应1~2h,TLC跟踪反应结束(PMA显色)。反应毕,旋去溶剂,DCM复溶,1N HCl水溶液洗涤2次,干燥,硅胶柱层析得5.2g淡黄色固体粗产品。
第三步:中间体3
在500mL圆底烧瓶中加入5克中间体2,用干燥的100mL MeOH溶解,0℃下加入4.2克NiCl2·6H2O,15min后分批加入2.7克NaBH4,30min后升至r.t.搅拌反应4h,TLC跟踪反应结束(PMA显色)。反应毕,抽滤除去滤渣,滤液旋干后得5g白色固体产品。
第四步:实施例1
在圆底烧瓶中,将0.055克中间体3、0.041克3-(4-甲基哌嗪-1-基)-丙酸、0.046克EDC、0.084mL三乙胺和0.003克DMAP溶于2mL二氯甲烷,常温反应2h,检测反应。反应毕,饱和NH4Cl水溶液洗涤2次,干燥,硅胶柱层析得25mg产品。
核磁数据为:1HNMR(400MHz,CDCl3)δ0.76(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);质谱:[M+1]570.5。
实施例1A
实施例1A的制备过程具体如下:
第一步:中间体4
在氩气保护下,将0.5克知母皂苷元原料A、0.4克对硝基苯甲酸、0.63克PPh3溶于干燥的5mL THF,冰水浴中搅拌5min,再缓慢滴加DIAD,搅拌10min,撤掉冰浴,常温反应3h,检测反应。反应完全后,旋干溶剂,用碳酸氢钠溶液/二氯甲烷萃取,过硅胶柱纯化PE:EA=45:1,点板检测(PE:EA=15:1),香草醛显色。产率55%。
第二步:中间体5
在0.36克中间体4和0.35克K2CO3中加入15mL MeOH,温度55℃,搅拌过夜。旋干溶剂,用水/二氯甲烷萃取,将有机层旋干得产物,直接投下一步反应,香草醛显色。产率80%。
第三步:中间体6
在氩气保护下,将0.2克中间体5、0.14克邻苯二甲酰亚胺、0.25克PPh3溶于干燥的4mL THF,冰水浴中搅拌5min,再缓慢滴加0.19克DIAD,搅拌10min,撤掉冰浴,常温反应3h,检测反应。旋干溶剂,用水/二氯甲烷萃取,过硅胶柱纯化PE:EA=45:1,爬板PE:EA=15:1,香草醛显色,产率50%。
第四步:中间体7
在0.13克中间体6和0.072克N2H4·H2O中加入10mL MeOH,温度55℃,搅拌过夜。旋干溶剂,用水洗,二氯甲烷萃取,将有机层旋干得产物,直接投下一步应,香草醛显色。产率90%。
第五步:实施例1A
在氩气保护下,将0.07克中间体、0.058克3-(4-甲基哌嗪-1-基)-丙酸、0.065克EDC和0.01克DMAP溶于4mL二氯甲烷,常温反应4h,检测反应。用碳酸氢钠溶液/二氯甲烷萃取,过碱性氧化铝柱PE:EA=1:1。香草醛显色,产率65%。
核磁数据为:1H NMR(400MHz,CDCl3)δ8.85(d,J=6.9Hz,1H),4.41(dd,J=13.0,6.6Hz,1H),4.18(s,1H),3.95(d,J=10.6Hz,1H),3.30(d,J=11.6Hz,1H),3.01-2.20(brs,8H),2.62(d,J=3.0Hz,2H),2.39(s,2H),2.30(s,3H),1.23-2.07(m,27H),1.08(d,J=6.7Hz,3H),1.05–0.93(m,6H),0.76(s,3H)。质谱:[M+1]570.5。
实施例1B
实施例1B的制备过程具体如下:
第一步:中间体8
以原料A为起始原料,通过与实施例1A中第三步同样的条件下,获得中间体8,产率50%。
第二步:中间体9
在该步骤中,以中间体8为原料,通过与实施例1A中第四步同样的条件,获得中间体9,产率90%。
第三步:实施例1B
在该步骤中,以中间体9和3-(4-甲基哌嗪-1-基)-丙酸为原料,通过与实施例1A中第五步同样的条件,获得实施例1B,产率65%。
核磁数据为:1H NMR(400MHz,CDCl3)δ8.15(d,J=7.6Hz,1H),4.33(dd,J=14.8,7.5 Hz,1H),3.88(dd,J=10.9,2.4Hz,1H),3.65(m,1H),3.23(d,J=10.9Hz,1H),2.90-2.10(brs,8H),2.56(t,J=6.2Hz,2H),2.26(m,2H),2.24(s,3H),1.23-2.07(m,27H),1.01(d,J=7.1Hz,3H),0.95–0.83(m,6H),0.69(s,3H)。质谱:[M+1]570.5。
实施例2
以中间体3和1-甲基哌啶-4-甲酸为原料,通过与实施例1中第四步描述的实验步骤相同的条件,获得实施例2。
核磁数据为:1HNMR(CDCl3,400MHz):δ0.75(s,3H),0.80-2.70(m,43H),2.95-4.00(m,7H),4.40-4.55(m,1H),6.05(s,1H);质谱:[M+1]541.5。
实施例3
以中间体3和N,N-二甲基甘氨酸为原料,通过与实施例1中第四步描述的实验步骤相同的条件,获得实施例3。
核磁数据为:1HNMR(CDCl3,400MHz):δ0.76(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);质谱:[M+1]501.3。
实施例4
以中间体3和N,N-二甲基-Β-丙氨酸为原料,通过与实施例1中第四步描述的实验步骤相同的条件,获得实施例4。
核磁数据为:1HNMR(CDCl3,400MHz):δ0.76(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)。质谱:[M+1]515.3。
实施例4A
以中间体7和N,N-二甲基-Β-丙氨酸为原料,通过与实施例1A中第五步描述的实验步骤相同的条件,获得实施例4A。
核磁数据为:1H NMR(400MHz,CDCl3)δ9.20(d,J=7.8Hz,1H),4.41(dd,J=14.1,7.6Hz,1H),4.19(d,J=7.2Hz,1H),3.95(dd,J=11.0,2.5Hz,1H),3.30(d,J=10.9Hz,1H),2.59–2.50(m,2H),2.38–2.33(m,2H),2.30(s,6H),1.23-2.07(m,27H),1.08(d,J=7.1Hz,3H),1.02–0.92(m,6H),0.76(s,3H)。质谱:[M+1]515.3。
实施例4B
以中间体9和N,N-二甲基-Β-丙氨酸为原料,通过与实施例1A中第五步描述的实验步骤相同的条件,获得实施例4B。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.77(d,J=7.2Hz,1H),4.41(dd,J=14.1,7.5Hz,1H),3.95(dd,J=10.9,2.3Hz,1H),3.78–3.65(m,1H),3.30(d,J=11.0Hz,1H),2.53(t,J=6.2Hz,2H),2.32(t,J=6.2Hz,2H),2.27(s,6H),1.23-2.07(m,27H),1.08(d,J=7.1Hz,3H),1.02–0.93(m,6H),0.76(s,3H)。质谱:[M+1]515.3。
实施例5
以中间体3和3-oxo-3-(4-甲基哌嗪-1-基)丙酸为原料,通过与实施例1中第四步描述的实验步骤相同的条件,获得实施例5。
核磁数据为:1HNMR(CDCl3,400MHz):δ0.76(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);质谱:[M+1]584.3。
实施例6
以中间体3和3-oxo-3-(1-甲基哌嗪-4-氨基)丙酸为原料,通过与实施例1中第四步描述的实验步骤相同的条件,获得实施例6。
核磁数据为:1HNMR(CDCl3,400MHz):δ0.76(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);质谱:[M+1]598.3。
实施例7
以中间体3和3-oxo-3-(1-甲基哌啶-4-甲基氨基)丙酸为原料,通过与实施例1中第四步描述的实验步骤相同的条件,获得实施例7。
核磁数据为:1HNMR(CDCl3,400MHz):δ0.76(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);质谱:[M+1]612.3。
实施例8
以中间体3和3-oxo-3-(3-吗啉丙基)氨基丙酸为原料,通过与实施例1中第四步描述的实验步骤相同的条件,获得实施例8。
核磁数据为:1HNMR(CDCl3,400MHz):δ0.75(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);质谱:[M+1]628.3。
实施例9
以中间体3和3-oxo-3-(1,4-双哌啶-1-)丙酸为原料,通过与实施例1第四步中描述的实验步骤相同的条件,获得实施例9。
核磁数据为:1HNMR(CDCl3,400MHz,ppm):δ0.76(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);质谱:[M+1]652.3。
实施例10
以中间体3和3-oxo-3-(哌嗪-1-)丙酸为原料,通过与实施例1中第四步描述的实验步骤相同的条件,获得实施例10。
核磁数据为:1HNMR(CDCl3,400MHz):δ0.77(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);质谱:[M+1]570.3。
实施例11
以中间体3和3-(六氢吡咯并[3,4-c]吡咯-2(1H)-基)-3-氧代丙酸(英文名称:3-(hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-oxopropanoic acid)为原料,通过与实施例1中第四步描述的实验步骤相同的条件,获得实施例11。
核磁数据为:1HNMR(CDCl3,400MHz):δ0.76(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);质谱:[M+1]596.4。
实施例12
以中间体3和3-氧-3-(4-(2-(吡喏烷-1-基)乙基)哌嗪-1-基)丙酸(英文名称:3-oxo-3-(4-(2-(pyrrolidin-1-yl)ethyl)piperazin-1-yl)propanoic acid)为原料,通过与实施例1中第四步描述的实验步骤相同的条件,获得实施例12。
核磁数据为:1HNMR(CDCl3,400MHz):δ0.74(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);质谱:[M+1]667.5。
实施例13
实施例13的制备过程具体如下:
第一步:中间体10
在500mL圆底烧瓶中加入中间体12(5克,1当量)、丙二酸单叔丁酯(2当量)、EDC·HCl(2当量)、DMAP(0.1当量)和Et3N(5当量),用100毫升干燥的DCM溶解,室温下搅拌反应2h,TLC跟踪反应结束(PMA显色)。反应毕,饱和NH4Cl水溶液洗涤2次,干燥,硅胶柱层析得4.5g产品。
第二步:中间体11
在500mL圆底烧瓶中加入中间体10(4.5克,1当量),用100毫升干燥的DCM溶解,0℃下滴加TFA(10毫升,15当量),15min后升至室温反应1~2h,TLC跟踪反应结束(PMA显色)。反应毕,旋干,DCM复溶,饱和NaHCO3水溶液洗涤2次,干燥,硅胶柱层析得4g产品白色固体即中间体11。
第三步:实施例13
在25mL圆底烧瓶中加入中间体11(50mg,1当量)、中间体12(2当量)、HATU(2当量)和Et3N(5当量),用3毫升干燥的DCM溶解,室温下搅拌反应1h,TLC跟踪反应结束(PMA显色)。反应毕,饱和NH4Cl水溶液洗涤2次,干燥,薄层板层析(二氯甲烷:甲醇=10:1)得8mg为白色固体。
核磁数据如下:1HNMR(CDCl3,400MHz,ppm):δ0.76(s,3H),δ0.80-2.30(m,36H),δ2.55-3.00(m,9H),δ3.15-4.50(m,14H),δ5.25(br s,1H);质谱:[M+1]641.5。
实施例13A
第一步 中间体13
原料A(460g,1.10mol,1.0eq)溶于吡啶(6.9L)中降温至0±5℃,将TsCl(210.5g,1.10mol,1.0eq)溶于吡啶(600mL)后,N2保护下缓慢加入反应液中,反应液自然升温至室温(23~28℃);搅拌4h后,室温N2保护下向反应液中补加105g TsCl(0.55mol,0.5eq)的吡啶(200mL)溶液,滴加完毕后室温(23~28℃)反应过夜,系统呈现酒红色,室温(23~28℃)搅拌48小时。反应完成后,将反应液缓慢滴入到0~10℃的水(65L)中,有大量固体析出,过滤,滤饼用PE:EA(50:1)混合溶剂500mL淋洗,所得固体在真空下(40~50℃水浴)干燥8小时得到451g固体,即中间体13。
第二步 中间体14
向一个500毫升烧瓶中,加入中间体13(25g,43.8mmol),醋酸钾(8.60g,87.6mmol)和18-冠-6(23.15g,87.6mmol)以及300毫升二甲亚砜溶剂。将反应体系升温至55摄氏度,保持此温度反应16小时。TLC监测反应(石油醚:乙酸乙酯=15:1)直至完全反应。然后,将反应混合物倒入1升冰水中,并搅拌30分钟。过滤,并用水洗涤滤饼,得7克白色固体。
第三步 中间体5
向一个250毫升烧瓶中,加入中间体14(2.0g,4.36mmol)以及60毫升四氢呋喃、60毫升甲醇、30毫升水以及5.5毫升4N的LiOH水溶液。将反应体系升温至60摄氏度,保持此温度反应2小时。用TLC板监测反应,展开体系为石油醚:乙酸乙酯=7:1。待反应完全后,旋去大部分有机溶剂,并加入50毫升水。过滤,并用水洗涤滤饼,得1.7克白色固体。
第四步 中间体15
向一个100毫升的反应烧瓶中,加入中间体5(5.0g,12.0mmol),TsCl(11.4g,60.0mmol)及DMAP(73mg,0.6mmol),并加入50毫升吡啶溶解。将反应体系升温至50摄氏度,并保持此温度反应16小时。TLC监测反应,展开剂为PE:EA=7:1。待反应完全后,将反应液倒入150毫升水,并搅拌30分钟。过滤反应液,并用水洗涤滤饼,得到5.5克白色固体。
第五步 中间体16
在30~35℃下向15(0.3g,0.52mmol,1eq)的DMF(6mL)悬浊液中依次滴加TMSN3(0.12g,1.1eq,2eq)和DBU(0.32g,0.21mmol,4eq),N2。所得反应液加热至80~90℃下反应16~20小时。TLC检测5基本消失(PE:EA=10:1)后,将反应液降温至30~35℃。将反应液导入到0~10℃的水(20mL)中,保持0~10℃搅拌5~10分钟后,过滤。滤饼经硅胶柱层析纯化PE:EA洗脱,得到0.13g固体即中间体16,收率56%。
第六步 中间体7
氮气保护下,向一个烧瓶中,加入中间体16(12.0g,27.2mmol)以及100毫升二氯甲烷溶剂。并加入4.8克Pd/C,以及200毫升乙醇。置换氢气(50psi)后,将反应体系升温至50摄氏度,并保持此温度反应16小时。用TLC监测反应,展开剂为石油醚:乙酸乙酯=20:1。过滤反应液,滤饼过柱(二氯甲烷:甲醇=20:1)得11克白色固体即中间体7。
第七步 中间体17
以中间体7为原料,按照中间体10描述的相同的条件,获得中间体17共40.4g。
第八步 中间体18
以中间体17为原料,按照中间体11描述相同的条件,获得中间体将18(35g)。
第九步 实施例13A
中间体18(28g,1.0eq)溶于DCM(300mL)中,加入中间体12(13.1g,1.5eq)、 T3P(35.3g,2.0eq)和NMM(11.2g,2.0eq),室温反应16h。TLC点板检测(DCM/MeOH(5%的氨甲醇)=20/1),反应完成后,反应液用(250mL)水洗,水相用DCM(100mL x 2)萃取,合并的有机相用无水Na2SO4干燥,过滤浓缩后过柱纯化(DCM/MeOH(5%的氨甲醇)=45/1~15/1)得实施例13A粗品约17克,将此粗品溶解于约二氯甲烷(120mL),然后加乙腈(200mL),将此溶液减压浓缩至约100毫升后于冰浴下搅拌2小时,析出大量白色固体,将反应物过滤,滤饼干燥再次放于室温的纯水(150mL)中打浆搅拌过夜,然后再过滤,干燥得实施例13A(12.2g)。
核磁数据为:1H NMR(400MHz,Chloroform-d)δ8.01(d,J=7.7Hz,1H),4.41(td,J=7.8,5.9Hz,1H),4.18(s,1H),3.95(dd,J=11.0,2.8Hz,1H),3.66(d,J=5.7Hz,2H),3.57(t,J=5.1Hz,2H),3.32–3.30(m,1H),3.29(d,J=3.7Hz,2H),2.55(s,4H),2.51–2.44(m,4H),2.37(m,6H),2.10–1.66(m,12H),1.66–1.45(m,7H),1.33–1.10(m,8H),1.08(m,3H),1.03–0.95(m,6H),0.75(s,3H)。质谱:[M+1]641.5。
实施例13B
第一步 中间体19
将中间体13(240g,0.42mol,1eq)和15-冠-5(277.8g,1.2mol,3eq)的DMSO(12L)混合物搅拌均匀至全部溶解,25~35℃,N2保护下向其中加入NaN3(81.9g,1.26mol,3eq)。所得混合物加热升温至60~70℃下反应2.5~3小时,N2保护,取样,TLC检测(PE:EA=9:1),原料消失,反应液温度降低至20~30℃后,加水,析出固体,过滤,滤饼用2L水淋洗干燥得到白色固体177g中间体19。
第二步 中间体23
将中间体19(650g)溶解在10L的DCM和10L的MeOH中,将溶液转入高压釜中。将104g 10%湿Pd/C悬浮在200mL MeOH中,转入高压釜中,28~35℃下,高压釜置换N2 4次,然后,28~35℃下,高压釜置换H2 4次。最后维持H2压力2.5~3MPa,然后,保持28~35℃下搅拌48h,取样TLC(PE:EA=20:1)检测原料消失。反应结束后在抽滤漏斗上铺一层硅藻土,减压抽滤,去除反应液中的Pd/C,滤饼用柱层析纯化得到白色固体中间体9共460g。
第三步 中间体20
以中间体9为原料,按照中间体10描述的相同实验步骤,获得中间体20共40.4g。
第四步 中间体21
以中间体20为原料,按照中间体11描述的相同实验步骤得粗品中间体21(35g)。
第五步 实施例13B
中间体21(28g,1.0eq)溶于DCM(300mL)中,加入中间体12(13.1g,1.5eq)、T3P(35.3g,2.0eq)和NMM(11.2g,2.0eq),室温反应16h。TLC点板检测(DCM/MeOH(5%的氨甲醇)=20/1),反应完成后,加250mL水,水相用DCM(100mL x 2)萃取,合并的有机相用无水Na2SO4干燥,过滤浓缩后过柱纯化(DCM/MeOH(5%的氨甲醇)=45/1~15/1)得实施例13B(12.2g)。
核磁数据:1H NMR(400MHz,Chloroform-d)δ7.39(d,J=8.2Hz,1H),4.42(td,J=7.6,6.0Hz,1H),3.96(dd,J=11.0,2.8Hz,1H),3.87–3.70(m,1H),3.65(t,J=5.2Hz,2H),3.56(q,J=5.2Hz,2H),3.31(d,J=11.0Hz,1H),3.28(s,2H),2.59–2.36(m,8H),2.28(s,6H),2.13–1.75(m,11H),1.75–1.49(m,7H),1.49–1.30(m,7H),1.30–1.11(m,7H),1.08(d,J=7.1Hz,4H),1.04(d,J=3.3Hz,1H),1.00(d,J=6.5Hz,3H),0.94(s,3H),0.75(s,3H)。质谱:[M+1]641.5。
实施例14
第一步:实施例14
在25mL圆底烧瓶中加入中间体11(50mg,1eq)、氨基乙醇(2eq)、HATU(2eq)和Et3N(5eq),用3毫升干燥的DCM溶解,室温下搅拌反应1h,TLC跟踪反应结束(PMA显色)。反应毕,饱和NH4Cl水溶液洗涤2次,干燥,薄层板层析(二氯甲烷:甲醇=10:1)得38mg实施例14为白色固体。
核磁数据如下: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);质谱:[M+1]545.5
实施例14A
以中间体18,用实施例14同样的合成方法可以得到白色固体实施例14A。
核磁数据为:1H NMR(400MHz,Chloroform-d)δ7.21(s,1H),7.07(d,J=7.7Hz,1H),4.41(q,J=7.5Hz,1H),4.18(s,1H),3.95(dd,J=11.0,2.8Hz,1H),3.74(q,J=5.1Hz,2H),3.45(td,J =5.6,4.5Hz,2H),3.30(d,J=11.0Hz,1H),3.18(s,2H),2.54(t,J=5.4Hz,1H),2.09-1.60(m,11H),1.53-1.11(m,14H),1.08(d,J=7.1Hz,4H),1.03-0.96(m,5H),0.76(s,3H)。质谱:[M+1]545.5
实施例14B
以中间体21,用实施例14同样的合成方法可以得到白色固体实施例14B。
核磁数据为:1H NMR(400MHz,Chloroform-d)δ7.42(s,1H),6.67(s,1H),4.41(td,J=7.8,6.2Hz,1H),3.96(dd,J=11.0,2.8Hz,1H),3.84-3.66(m,3H),3.44(td,J=5.6,4.4Hz,2H),3.35-3.22(m,1H),3.16(s,2H),2.81(s,1H),2.00(m,2H),1.92-1.75(m,5H),1.75-1.50(m,9H),1.50-1.31(m,7H),1.31-1.13(m,6H),1.08(m,3H),1.00(d,J=6.6Hz,3H),0.95(s,3H),0.75(s,3H)。质谱:[M+1]545.5
实施例15
以异知母菝契皂苷元为原料,通过与实施例14相同方法,获得实施例15。
核磁数据:1H NMR(400MHz,Chloroform-d)δ7.48(s,1H),7.40(d,J=7.0Hz,1H),6.82(s,1H),4.47-4.33(m,1H),4.17(s,1H),3.74(m,2H),3.52-3.41(m,3H),3.38(m,1H),3.19(d,J=14.7Hz,2H),3.07(q,J=7.4Hz,2H),2.08-1.94(m,2H),1.94-1.81(m,2H),1.81-1.66(m,4H),1.60(dd,J=16.2,11.9Hz,5H),1.56-1.41(m,5H),1.31-1.10(m,6H),1.10-1.02(m,2H),1.00(s,1H),0.99-0.92(m,4H),0.79(dd,J=6.3,1.5Hz,3H),0.75(d,J=1.8Hz,3H)。质谱:[M+1]545.5
实施例15A
以异知母菝契皂苷元为原料,通过与实施例14A相同方法,获得实施例15A。
核磁数据:1H NMR(400MHz,Chloroform-d)δ7.40(s,1H),7.30(d,J=7.6Hz,1H),4.40(td,J=7.7,5.9Hz,1H),4.17(s,1H),3.73(d,J=4.8Hz,2H),3.41(m,4H),3.20(s,2H),2.92(s,1H),1.43(m,33H),0.78(m,6H)。质谱:[M+1]545.5
实施例15B
以异知母菝契皂苷元为原料,通过与实施例14A相同方法,获得实施例15B。
核磁数据:1H NMR(400MHz,Chloroform-d)δ7.51(s,1H),6.83(d,J=7.9Hz,1H),4.41(q,J=7.4Hz,1H),3.74(m,3H),3.42(m,4H),3.17(s,2H),1.38(m,34H),0.78(m,6H)。质谱:[M+1]545.5
实施例16A
以异知母菝契皂苷元为原料,通过与实施例13A相同方法,获得实施例16A。
核磁数据为:1H NMR(400MHz,Chloroform-d)δ8.02(d,J=7.7Hz,1H),4.46-4.33(m,1H),4.18(d,J=7.3Hz,1H),3.66(dq,J=5.7,3.1,2.2Hz,2H),3.57(q,J=5.2,4.6Hz,2H),3.47(ddd,J=10.9,4.4,2.0Hz,1H),3.37(t,J=10.9Hz,1H),3.30(d,J=3.4Hz,2H),2.52(d,J=2.9Hz,4H),2.48(t,J=6.1Hz,4H),2.33(d,J=6.0Hz,7H),2.07-1.28(m,29H),1.23-1.02(m,10H),1.00(s,3H),0.96(d,J=6.9Hz,4H),0.92-0.81(m,5H),0.79(d,J=6.3Hz,3H),0.76(s,3H)。质谱:[M+1]641.5。
实施例16B
以异知母菝契皂苷元为原料,通过与实施例13B相同的制备步骤及相同的条件,获得实施例16B。
核磁数据为:1H NMR(400MHz,Chloroform-d)δ7.40(d,J=8.1Hz,1H),4.49-4.29(m,1H),3.88-3.70(m,1H),3.65(t,J=5.1Hz,2H),3.60-3.53(m,2H),3.53-3.45(m,1H),3.38(t,J=10.9Hz,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=6.3Hz,3H),0.75(s,3H)。质谱:[M+1]641.5。
实施例17A
以异知母皂苷元为原料,通过与中间体7相同的制备步骤及相同的条件,获得中间体22;以中间体22为原料,通过与实施例1A步骤五描述的相同制备方法以及条件,获得实施例17A。质谱:[M+1]570。
实施例17B
以异知母皂苷元为原料,通过与中间体9相同的制备步骤及相同的条件,获得中间体23;以中间体23为原料,通过与实施例1B描述的相同制备方法以及条件,获得实施例17B。
质谱:[M+1]570。
实施例18A
以异知母皂苷元为原料,通过与实施例4A描述的相同制备步骤及条件,获得实施例18A。
核磁数据为:1H NMR(400MHz,Chloroform-d)δ9.14(s,1H),4.45-4.34(m,1H),4.19(d,J=8.3Hz,1H),3.52-3.42(m,2H),3.37(t,J=10.9Hz,1H),2.56(t,J=5.9Hz,2H),2.36(dd,J=6.5,5.2Hz,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=6.3Hz,3H),0.76(s,3H)。质谱:[M+1]515。实施例18B
以异知母皂苷元为原料,通过与实施例4B描述的相同制备步骤及条件,获得实施例18B。
核磁数据为:1H NMR(400MHz,Chloroform-d)δ6.46(s,1H),4.46-4.34(m,1H),3.81-3.60(m,1H),3.47(d,J=4.6Hz,1H),3.38(t,J=10.9Hz,1H),3.26(t,J=6.6Hz,2H),2.80(t,J=6.6Hz,2H),2.72(s,6H),2.05-1.53(m,16H),1.46(d,J=4.3Hz,2H),1.37-1.33(m,2H),1.19-1.00(m,9H),0.99-0.91(m,7H),0.91-0.81(m,14H),0.79(d,J=6.3Hz,3H),0.75(s,3H)。质谱:[M+1]515。
实施例19A
以海柯皂苷元为原料,通过与中间体7相同的制备步骤及相同的条件,获得中间体24;以中间体24为原料,通过与实施例1A步骤五描述的相同制备方法以及条件,获得实施例19A。
核磁数据为:1H NMR(400MHz,Chloroform-d)δ4.33(t,J=7.4Hz,1H),3.79-3.61(m,3H),3.50(s,2H),3.35(t,J=10.9Hz,2H),3.10(q,J=7.4Hz,2H),2.99(s,7H),2.70(s,1H),2.62-2.45(m,5H),2.38(t,J=13.7Hz,2H),2.22(dd,J=14.3,5.0Hz,2H),2.11(q,J=6.5,6.0Hz,2H),1.90(d,J=9.0Hz,3H),1.75(dt,J=12.9,6.5Hz,5H),1.69-1.50(m,11H),1.44(dd,J=11.3,7.9Hz,6H),1.40-1.09(m,10H),1.09-1.01(m,5H),0.97(dd,J=12.9,7.9Hz,2H),0.89(s,4H),0.79(d,J=6.3Hz,3H)。质谱:[M+1]529.4。
实施例20A
以海柯皂苷元为原料,通过与实施例4A描述的相同制备步骤及条件,获得实施例20A。
核磁数据为:1H NMR(400MHz,Chloroform-d)δ6.99(t,J=6.4Hz,1H),4.33(dd,J=8.5,5.8Hz,1H),3.81-3.57(m,1H),3.57-3.43(m,1H),3.35(t,J=11.0Hz,1H),3.15(t,J=6.5Hz,2H),2.71(t,J=6.5Hz,2H),2.66(s,6H),2.51(dd,J=8.8,6.7Hz,1H),2.38(t,J=13.7Hz,1H),2.21(dd,J=14.3,5.0Hz,1H),2.11(t,J=7.1Hz,1H),1.89(dd,J=11.1,7.3Hz,1H),1.83-1.71(m,3H),1.71-1.52(m,6H),1.52-1.38(m,3H),1.38-1.09(m,7H),1.09-0.99(m,6H),0.89(s,3H),0.79(d,J=6.3Hz,3H)。质谱:[M+1]529.4。
实施例21
以4-羟基苯乙胺为原料,通过与实施例13描述的相同制备步骤及条件,获得实施例21。
核磁数据为:1HNMR(CDCl3,40MHz,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);质谱:[M+1]621.4
实施例22
以3,4-二羟基苯乙胺为原料,通过与实施例13描述相同的制备步骤及条件,获得实施例22。
核磁数据如下: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);质谱:[M+1]637.4
实施例23
其制备过程具体如下:
第一步:中间体25
在氩气保护下,将0.2克吗啉、0.4克1-Boc-3-氮杂环丁酮、0.7mL醋酸、0.3克氰基硼氢化钠溶于20mL二氯甲烷,常温反应2h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率70%。
第二步:中间体26
在氩气保护下,将0.2克中间体25溶于10mL二氯甲烷,加入5mL盐酸二氧六环溶液,常温反应2h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率90%。
第三步:实施例23的合成
在氩气保护下,将0.1克中间体26、0.43克中间体11、0.40克HATU和0.22克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率60%。
核磁数据为:1H NMR(400MHz,CD3OD)δ4.5-4.65(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)。质谱:[M+1]626.5。
实施例24
其制备过程具体如下:
在氩气保护下,将0.2克中间体11、0.08克1-甲基-4-哌啶-4-基哌嗪、0.227克HATU和0.101克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,香草醛显色,产率45%。
核磁数据为:1H NMR(400MHz,CDCl3)δ8.13(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)。质谱:[M+1]667.5。
实施例24A,实施例24B
以中间体18,用实施例24同样的合成方法可以得到实施例24A。以中间体21,同样的方法得到实施例24B。
实施例24A核磁数据为:1H NMR(400MHz,CDCl3)δ8.12(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).质谱:[M+1]667.5。
实施例24B核磁数据为:1H NMR(400MHz,CDCl3)δ7.42(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).质谱:[M+1]667.5。
实施例25
其制备过程具体如下:
其制备方法与实施例24一样。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.75(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)。质谱:[M+1]681.6。
实施例25A、实施例25B
以中间体18和1-乙基-4-(哌啶-4-甲基)哌嗪,用实施例24同样的合成方法可以得到实施例25A。以中间体21和1-乙基-4-(哌啶-4-甲基)哌嗪,同样的方法得到实施例25B。
实施例25A核磁数据为:1H NMR(400MHz,CDCl3)δ8.15(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).质谱:[M+1]681.6。
实施例25B核磁数据为:1H NMR(400MHz,CDCl3)δ7.38(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).质谱:[M+1]681.6。
实施例26
其制备过程具体如下:
其制备方法与实施例24一样。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.74(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).质谱:[M+1]667.6。实施例26A,实施例26B
以中间体18和1-(1-甲基-4-哌啶)哌嗪,用实施例24同样的合成方法可以得到实施例26A。以中间体21和1-(1-甲基-4-哌啶)哌嗪,同样的方法得到实施例26B。
实施例26A核磁数据为:1H NMR(400MHz,CDCl3)δ8.09(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).质谱:[M+1]667.6。
实施例26B磁数据为:1H NMR(400MHz,CDCl3)δ7.38(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).质谱:[M+1]667.6。
实施例27
其制备过程具体如下:
第一步:中间体27
将0.5克4-(2-氨基乙基)哌嗪-1-羧酸叔丁酯、0.714克二碳酸二叔丁酯溶于干燥的20mL DCM,常温反应12h,检测反应。反应完全后,旋干溶剂,用水/二氯甲烷萃取,过硅胶柱纯化DCM:MeOH=30:1,点板检测(DCM:MeOH=10:1),香草醛显色。产率65%。
第二步:中间体28
在0.2克中间体27和0.07克LiAlH4中加入15mL THF,加热至回流状态,反应3h。检测反应。反应完全后,冷却至室温,旋干溶剂得产物,直接投下一步反应,香草醛显色。产率56%。
第三步:实施例27的合成
在氩气保护下,将0.037克中间体28、0.12克中间体11、0.136克HATU和0.061克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,香草醛显色,产率55%。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.97(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).。质谱:[M+1]641.6。
实施例27A,实施例27B
其合成同实施例27,具体如下
以中间体18和N-甲基-2-(1-甲基哌啶-4-基)乙烷-1-胺,用实施例27同样的合成方法可以得到实施例27A。以中间体21和N-甲基-2-(1-甲基哌啶-4-基)乙烷-1-胺,同样的方法得到实施例27B。
实施例27A核磁数据为:1H NMR(400MHz,CDCl3)δ8.45(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).质谱:[M+1]641.6。
实施例27B核磁数据为:1H NMR(400MHz,CDCl3)δ8.7.78(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).质谱:[M+1]641.6。
实施例28
其制备过程具体如下:
在氩气保护下,将0.1克哌嗪-1-羧酸甲酯、0.42克中间体11、0.40克HATU和0.21克 TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率65%。
核磁数据为:1H NMR(CDCl3-d6,400MHz):7.12(d,1H),4.35-4.46(m,1H),3.90-4.01(m,1H),3.70-3.85(m,1H),3.73(s,3H),3.60-3.68(m,2H),3.45-3.58(m,6H),3.28-3.35(m,1H),3.29(s,2H),1.58-2.10(m,11H),1.15-1.50(m,16H),1.08(d,3H),0.99(d,3H),0.95(s,3H),0.75(s,3H).质谱:[M+1]628.5。
实施例29
其制备过程具体如下:
第一步:中间体29
在氩气保护下,将0.1克1-Boc-4-(哌啶-4-基)-哌嗪、0.05克溴乙烷、0.08克TEA溶于30mL二氯甲烷,常温反应12h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率80%。
第二步:中间体30
在氩气保护下,将0.1克中间体29溶于10mL二氯甲烷,加入5mL盐酸二氧六环溶液,常温反应2h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率90%。
第三步:实施例29的合成
在氩气保护下,将0.05克中间体30、0.15克中间体11、0.15克HATU和0.08克TEA 溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率60%。
核磁数据为:1H NMR(400MHz,CDCl3)δ8.7.38(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).质谱:[M+1]681.5。
实施例29A,实施例29B
其合成过程同实施例29以中间体18和1-(1-乙基哌啶-4-基)哌嗪,用实施例29同样的合成方法可以得到实施例29A。以中间体21和1-(1-乙基哌啶-4-基)哌嗪,同样的方法得到实施例29B。
实施例29A核磁数据为:1H NMR(400MHz,CDCl3)δ8.8.05(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)..质谱:[M+1]681.6。
实施例29B核磁数据为:1H NMR(400MHz,CDCl3)δ8.7.38(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)..质谱:[M+1]681.5。
实施例30
其制备过程具体如下:
第一步:中间体31
在氩气保护下,将0.4克中间体11、0.258克4-(哌啶-4-基)哌嗪-1-羧酸叔丁酯、0.455克HATU和0.202克TEA溶于15mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,香草醛显色,产率75%。
第二步:中间体32
在0.2克中间体31加入10mL MeOH,再加入10mL的4M的HCl/dioxane溶液,室温反应3h。旋干溶剂,直接投下一步反应,香草醛显色。产率80%。
第三步:实施例30的合成
在氩气保护下,将0.08克中间体32、0.03克溴乙腈、0.034克碳酸钾和0.02克碘化钾溶于10mL乙腈,升温到50℃,反应12h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,香草醛显色,产率65%。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.37(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).。质谱:[M+1]693.6。
实施例31
其制备过程具体如下:
第一步:中间体33
将0.1克4-(哌啶-4-基)哌嗪-1-羧酸叔丁酯、0.089克溴乙睛溶于干燥的10mL MeCN中,再加入0.103g的碳酸钾和0.062g的碘化钾,升温到50℃,反应12h,检测反应。反应完全后,用水/二氯甲烷萃取,过硅胶柱纯化DCM:MeOH=30:1,点板检测(DCM:MeOH=10:1),香草醛显色。产率55%。
第二步:中间体34
在0.2克中间体33加入10mL MeOH,再加入10mL的4M的HCl/dioxane溶液,室温反应3h。旋干溶剂,直接投下一步反应,香草醛显色。产率80%。
第三步:实施例31的合成
在氩气保护下,将0.046克中间体34、0.1克中间体11、0.114克HATU和0.061克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,香草醛显色,产率40%。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.35(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)。质谱:[M+1]692.6。
实施例32
其制备过程具体如下:
第一步:中间体35
在氩气保护下,将0.1克1-Boc-4-(哌啶-4-基)-哌嗪、0.06克溴代异丁烷、0.08克TEA溶于30mL N,N-二甲基甲酰胺,升温到50℃,反应12h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率70%。
第二步:中间体36
在氩气保护下,将0.1克中间体35溶于10mL二氯甲烷,加入5mL盐酸二氧六环溶液,常温反应2h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率90%。
第三步:实施例36的合成
在氩气保护下,将0.05克中间体36、0.11克中间体11、0.13克HATU和0.07克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率60%。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.32(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)..质谱:[M+1]709.7。
实施例32A、实施例32B
其合成方法同32
以中间体18和1-(1-异丁基哌啶-4-基)哌嗪,用实施例32同样的合成方法可以得到实施例32A。以中间体21和1-(1-异丁基哌啶-4-基)哌嗪,同样的方法得到实施例32B。
实施例32A核磁数据为:1H NMR(400MHz,CDCl3)δ7.32(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).质谱:[M+1]709.7。
实施例32B核磁数据为:1H NMR(400MHz,CDCl3)δ7.32(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).质谱:[M+1]709.7。
实施例33
其制备过程具体如下:
第一步:中间体37
在氩气保护下,将0.1克1-Boc-4-(哌啶-4-基)-哌嗪、0.22克中间体11、0.21克HATU和0.11克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率60%。
第二步:中间体38
在氩气保护下,将0.1克中间体37溶于10mL二氯甲烷,加入5mL盐酸二氧六环溶液,常温反应2h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率90%。
第三步:实施例33的合成
在氩气保护下,将0.1克中间体38、0.03克溴代异丁烷、0.03克TEA溶于15mL N,N-二甲基甲酰胺,升温到50℃,反应12h,检测反应。旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率60%。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.32(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)..质谱:[M+1]709.6。
实施例33A,实施例33B
其合成过程如下,以中间体18和1-溴-2-甲基丙烷,用实施例33同样的合成方法可以得到实施例33A。以中间体21和1-溴-2-甲基丙烷,同样的方法得到实施例33B。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.32(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)..质谱:[M+1]709.6。
实施例33B核磁数据为:1H NMR(400MHz,CDCl3)δ7.32(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)..质谱:[M+1]709.5。
实施例34
其制备过程具体如下:
第一步:中间体39
在氩气保护下,将0.1克1-Boc-4-(哌啶-4-基)-哌嗪、0.06克2-溴丙烷、0.08克TEA溶于30mL N,N-二甲基甲酰胺,升温到50℃,反应12h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率70%。
第二步:中间体40
在氩气保护下,将0.1克中间体39溶于10mL二氯甲烷,加入5mL盐酸二氧六环溶液,常温反应2h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率90%。
第三步:实施例34的合成
在氩气保护下,将0.05克中间体40、0.14克中间体11、0.13克HATU和0.07克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率60%。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.32(dd,1H),4.41-4.45(m,1H),3.94-3.98(m,1H),3.75-3.78(m,1H),3.51(m,6H),3.28-3.32(m,3H),2.55(m,8H),1.32-2.10(m,27H),1.19(m, 14H),1.08(m,6H),0.99(m,3H),0.75(s,3H).质谱:[M+1]695.7。
实施例34A,实施例34B
其合成过程如下,以中间体18和1-(1-异丙基哌啶-4-基)哌嗪,用实施例34同样的合成方法可以得到实施例34A。以中间体21和1-(1-异丙基哌啶-4-基)哌嗪,同样的方法得到实施例34B。
实施例34A核磁数据为:1H NMR(400MHz,CDCl3)δ7.33(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).质谱:[M+1]695.7。
实施例34B核磁数据为:1H NMR(400MHz,CDCl3)δ7.32(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).质谱:[M+1]695.6。
实施例35
其制备过程具体如下:
以中间体11和1-(吡啶-4-基)哌嗪为原料,用实施例28同样的合成方法可以得到实施例35
核磁数据为:1H NMR(400MHz,CDCl3)δ8.25(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).质谱:[M+1]647.5。
实施例35A,实施例35B
其合成过程如下,以中间体18和1-(吡啶-4-基)哌嗪,用实施例28同样的合成方法可以得到实施例35A。以中间体21和1-(吡啶-4-基)哌嗪,同样的方法得到实施例35B。
实施例35A核磁数据为:1H NMR(400MHz,CDCl3)δ8.24(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).质谱:[M+1]647.6。
实施例35B核磁数据为:1H NMR(400MHz,CDCl3)δ8.26(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).质谱:[M+1]647.5。
实施例36
其制备过程具体如下:
以中间体11和1-(1-(2-氟乙基)哌啶-4-基)哌嗪为原料,用实施例31同样的合成方法可以得到实施例36
核磁数据为:1H NMR(400MHz,CDCl3)δ8.09(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).质谱:[M+1]699.6。
实施例37
其制备过程具体如下:
第一步:中间体41
在氩气保护下,将0.2克N,N-二甲基甘氨酸、0.36克1-叔丁氧羰基哌嗪、1.1克HATU和0.6克TEA溶于60mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率80%。
第二步:中间体42
在氩气保护下,将0.2克中间体41溶于10mL二氯甲烷,加入5mL盐酸二氧六环溶液,常温反应2h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率90%。
第三步:实施例37的合成
在氩气保护下,将0.05克中间体42、0.18克中间体11、0.17克HATU和0.1克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率60%。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.76(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).质谱:[M+1]655.6。
实施例38
其制备过程具体如下:
第一步:中间体43
在氩气保护下,将0.1克1-Boc-4-(哌啶-4-基)-哌嗪、0.1克2-溴-N,N-二甲基乙酰胺、0.08克TEA溶于30mL二氯甲烷,常温反应12h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率80%。
第二步:中间体44
在氩气保护下,将0.1克中间体43溶于10mL二氯甲烷,加入5mL盐酸二氧六环溶液,常温反应2h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率90%。
第三步:实施例38的合成
在氩气保护下,将0.05克中间体44、0.18克中间体11、0.17克HATU和0.09克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率60%。
核磁数据为:1H NMR(400MHz,DMSO)δ8.18(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).质谱:[M+1]655.6。
实施例39
其制备过程具体如下:
第一步:中间体45
在氩气保护下,将0.1克N,N-二甲胺基溴乙烷氢溴酸盐、0.08克1,4-二氮杂环庚烷-1-甲酸叔丁酯、0.13克TEA溶于30mL二氯甲烷,常温反应12h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率80%。
第二步:中间体46
在氩气保护下,将0.1克中间体45溶于10mL二氯甲烷,加入5mL盐酸二氧六环溶液,常温反应2h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率90%。
第三步:实施例39的合成
在氩气保护下,将0.05克中间体46、0.18克中间体11、0.17克HATU和0.09克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率60%。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.90(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)..质谱:[M+1]655.6。
实施例40
其制备过程具体如下:
第一步:中间体47
在氩气保护下,将0.2克咪唑、0.5克1-溴-2-氯乙烷、0.6克TEA溶于30mL二氯甲烷,常温反应12h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率80%。
第二步:中间体48
在氩气保护下,将0.1克1-Boc-4-(哌啶-4-基)-哌嗪、0.08克中间体47、0.08克TEA溶于30mL二氯甲烷,常温反应12h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率70%。
第三步:中间体49
在氩气保护下,将0.1克中间体48溶于10mL二氯甲烷,加入5mL盐酸二氧六环溶液,常温反应2h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率90%。
第四步:实施例40的合成
在氩气保护下,将0.05克中间体49、0.17克中间体11、0.16克HATU和0.08克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率60%。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.90(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)..质谱:[M+1]664.6。
实施例41
其制备过程具体如下:
第一步:中间体50
在氩气保护下,将0.2克2-二乙氨基-1-溴乙烷氢溴酸盐、0.15克1-叔丁氧羰基哌嗪溶于40mL乙腈,50℃反应12h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率80%。
第二步:中间体51
在氩气保护下,将0.1克中间体50溶于10mL二氯甲烷,加入5mL盐酸乙酸乙酯溶液,常温反应12h,检测反应。旋干溶剂,直接投下一步反应,磷钼酸显色。产率90%。
第三步:实施例41的合成
在氩气保护下,将0.05克中间体51、0.14克中间体11、0.15克HATU和0.08克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,磷钼酸显色,产率60%。
核磁数据为:1H NMR(400MHz,CDCl3)δ7.32(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).质谱:[M+1]669.7。
实施例41A,实施例41B
实施例41A的合成过程如实施例41,中间体18代替中间体11,得到,核磁数据为1H NMR(CDCl3-d6,400MHz):7.98(d,1H),4.42(q,1H),4.18(s,1H),3.95(dd,1H),3.66(t,2H),3.57(t,2H),3.31(d,1H),3.29(s,2H),2.67-2.82(m,8H),2.47-2.52(m,4H),1.58-2.10(m,11H),1.15-1.50(m,16H),1.09-1.10(m,6H),1.08(d,3H),0.99(d,3H),0.95(s,3H),0.75(s,3H).质谱:[M+1]669.7。
实施例41B的合成过程如实施例41,中间体21代替中间体11,得到,核磁数据为1H NMR(CDCl3-d6,400MHz):7.35(d,1H),4.42(q,1H),3.98(dd,1H),3.73-3.78(m,1H),3.63(t,2H),3.56(t,2H),3.32(d,1H),3.28(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).质谱:[M+1]669.6。
实施例42
其制备过程具体如下
第一步:中间体52
在氩气保护下,将1克中间体3、0.933克3-(1-叔丁氧羰基哌嗪-4-YL)丙酸、1.373克HATU和0.788克DIPEA溶于20mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化DCM:MeOH=20:1,爬板DCM:MeOH=10:1,香草醛显色,产率65%。
第二步:中间体53
在1克中间体52溶于10mL MeOH中,加入4M的HCl/dioxane,反应3h。检测反应。反应完全后,旋干溶剂得产物,直接投下一步反应,香草醛显色。产率80%。
第三步:实施例42的合成
在氩气保护下,将0.2克中间体53、0.077克3-吡咯烷-1-基丙酸、0.205克HATU和0.091克TEA溶于10mL二氯甲烷,常温反应6h,检测反应。用水/二氯甲烷萃取,饱和食盐水洗涤,无水硫酸钠干燥,旋干溶剂,过硅胶柱纯化(DCM:MeOH=20:1),香草醛显色,产率60%。核磁数据为:1H NMR(400MHz,CDCl3)δ7.13(d,1H),4.42(q,1H),3.62(m,9H),2.89(d,2H),2.51(m,17H),1.31(m,38H).质谱:[M+1]682.6。
实施例42A
其制备过程与实施例42一样,用中间体7代替中间体3得到实施例42A。核磁数据为:1H NMR(400MHz,CDCl3)δ8.29(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).质谱:[M+1]682.7。
实施例43化合物活性测试
实验1.小分子化合物对糖氧剥夺模型(OGD)诱导大鼠原代培养神经元损伤的保护作用检测
实验与分析方法:订购孕15天的大鼠,解剖胎鼠大脑(E15-16),使用48孔板在神经元培养基中进行大脑皮层神经元原代培养14天(DIV 14),进行OGD实验。OGD实验过程中将神经元培养液换为无糖无氧培养液(95%N2/5%CO2平衡),神经元在OGD chamber中接受OGD处理。然后换为正常神经元培养液,放95%Air/5%CO2孵箱中培养24小时后进行神经元活性分析。通过Hoechst 33342和Propidium iodide染色,利用高内涵荧光显微镜拍照,每个重复孔拍摄20个随机不重合视野的照片,软件(Thermo ScientificTM,HCS Studio Cell Analysis Software)计数总神经元数和死亡神经元数目。利用所得到的数据计算标准差和标准误差,做T-test分析计算P值,确定是否有显著性差异。
化合物处理:将实施例1到实施例20中的13种小分子化合物用DMSO稀释至0.5mM母液,48孔板每孔0.5ml培养液中分别加入1μL母液至终浓度为1μM,阳性对照化合物1μL DPQ(母液10mM),阴性对照1μL DMSO在OGD实验前24小时加入培养液,并在无糖无氧培养液和后续正常神经元培养液中添加相同浓度。
结果:如图1所示,本次OGD实验结果中,未做OGD的神经元死细胞比例为10.32%,加DMSO的阴性对照死细胞比例为52.31%,损伤率为42%左右。加DPQ的阳性对照死细胞比例为22.22%,对神经元损伤的保护作用非常显著。
实验2.实施例1~实施例14B中的小分子化合物对过氧化氢(H2O2)诱导人SHSY5Y神经肿瘤细胞氧化损伤的保护作用检测
表1.实验条件及方法
表2.实验流程
结果:如图2所示,实施例2、实施例7、实施例13、实施例14A显著保护过氧化氢诱导的神经肿瘤细胞损伤,其中,未分化的SH-SY5Y细胞,预保护24h,终浓度1μM,过氧化氢处理24h后检测。
实验3.小分子化合物对L-半胱氨酸诱导的神经元兴奋性毒性的保护作用检测
实验与分析方法:实验前24孔细胞培养板用Poly-D-lysine预处理,放置于37℃,5%CO2的细胞培养箱内过夜。订购孕18天的SD大鼠,解剖胎鼠大脑(E18-19),使用24孔板在神经元培养基中进行大脑海马神经元原代培养,采用无血清培养基培养海马神经元。在37℃,5%CO2的细胞培养箱内培养第19天(DIV19)后加入待测化合物(终浓度1μM)、阳性对照AP5[(2R)-氨基-5-膦戊酸(酯)](终浓度100μM),培养24小时后加入L-Cystine(400μM)和NaHCO3(10mM)。再过18小时,加入hoechst3342(2.5μg/ml)进行细胞染色。放置于培养箱内细胞孵育15分钟,利用高内涵荧光显微镜拍照,每个重复孔拍摄6个随机不重合视野的照片,用软件Image J计数总神经元数目和死亡神经元数目。利用所得到的数据确定药物处理组与对照组是否有差异。
化合物处理:将实施例1到实施例19A中的32种小分子化合物用DMSO稀释至1mM母液,24孔板每孔1ml培养液中分别加入1μl母液至终浓度为1μM,阳性对照化合物100μl AP5(母液100mM),阴性对照1μl DMSO。
结果:如图3所示,未加入L-Cystine(400μM)和NaHCO3(10mM)的神经元死细胞比例为20.12%,加DMSO的阴性对照死细胞比例为87.62%,损伤率为67.5%左右。加AP5的阳性对照死细胞比例为27.58%,AP5对神经元兴奋性毒性的保护作用非常显著。加入上述4个小分子化合物的实验的结果显示,化合物实施例1,实施例1A,实施例2,实施例4B,实施例6,实施例10,实施例13,实施例13B等对L-Cystine(400μM)诱导的神经元兴奋性毒性具有保护作用。
实验4.“实施例5”和“实施例6”在斑马鱼上的抗炎作用评价
实验动物
转基因中性粒细胞荧光斑马鱼,以自然成对交配繁殖方式进行。年龄为受精后3天(3dpf),共810尾,每实验组为30尾。用于确定LPS诱发炎症实验中“实施例5”和“实施例6”最大检测浓度(MTC)以及“实施例5”和“实施例6”对LPS诱发炎症的抗炎作用评价。
斑马鱼饲养于28℃的养鱼用水中(水质:每1L反渗透水中加入200mg速溶海盐,电导率为480~510μS/cm;pH为6.9~7.2;硬度为53.7~71.6mg/L CaCO3),由本公司养鱼中心繁殖提供,实验动物使用许可证号为:SYXK(浙)2012-0171。饲养管理符合国际AAALAC认证的要求。
实验药物
“实施例5”,白色粉末,4℃干燥保存,于2019年10月18日收样,由深圳清博汇能医药科技有限公司提供。实验前用DMSO配制成20mM的母液,-20℃保存。
“实施例6”,白色粉末,4℃干燥保存,于2019年10月18日收样,由深圳清博汇能医药科技有限公司提供。实验前用DMSO配制成20mM的母液,-20℃保存。
吲哚美辛,批号为1108939,上海晶纯实业有限公司生产,由杭州环特生物科技股份有限公司提供。实验前用DMSO溶解配制得到80mM的母液,-20℃保存。
仪器和试剂
解剖显微镜(SZX7,OLYMPUS,Japan);与显微镜相连的相机(VertA1);精密电子天平(CP214,OHAUS,AmericaCP214,OHAUS);荧光显微镜(AZ100,Nikon,Japan);甲基纤维素(Sigma,USA);二甲基亚砜(Sigma,France);6孔板(Nest Biotech)。
浓度组别
1、确定“实施例5”和“实施例6”的最大检测浓度(MTC)
实验1组 正常对照组(不做任何处理)
实验2组 模型对照组
实验3组 “实施例5”0.625μM
实验4组 “实施例5”1.25μM
实验5组 “实施例5”2.5μM
实验6组 “实施例5”5μM
实验7组 “实施例5”10μM
实验8组 “实施例5”50μM
实验9组 “实施例5”100μM
实验10组 “实施例5”200μM
实验11组 “实施例6”0.625μM
实验12组 “实施例6”1.25μM
实验13组 “实施例6”2.5μM
实验14组 “实施例6”5μM
实验15组 “实施例6”10μM
实验16组 “实施例6”50μM
实验17组 “实施例6”100μM
实验18组 “实施例6”200μM
2、评价“实施例5”和“实施例6”抗炎作用
实验1组 正常对照组(不做任何处理)
实验2组 模型对照组
实验3组 阳性对照药吲哚美辛80μM
实验4组 “实施例5”0.28μM
实验5组 “实施例5”0.83μM
实验6组 “实施例5”2.5μM
实验7组 “实施例6”0.28μM
实验8组 “实施例6”0.83μM
实验9组 “实施例6”2.5μM
浓度确定依据
根据浓度摸索结果与客户沟通后,确定“实施例5”和“实施例6”抗炎作用评价的最大检测浓度均为2.5μM。
模型制作
LPS以卵黄囊注射给样方式处理正常3dpf转基因中性粒细胞荧光斑马鱼,建立斑马鱼炎症模型。
实验方法
1、确定“实施例5”和“实施例6”的最大检测浓度(MTC)
在显微镜下挑选发育健康且阶段一致的受精后3天的(3dpf)转基因中性粒细胞荧光斑马鱼于六孔板中,每孔随机挑选30尾,每孔容量为3mL,卵黄囊注射给予LPS建立斑马鱼炎症模型。分别水溶给予“实施例5”和“实施例6”,浓度均为0.625、1.25、2.5、5、10、50、100和200μM;同时设置正常对照组(即养鱼用水处理斑马鱼)与模型对照组,置于28℃培养箱中培养3小时后观察记录斑马鱼的死亡情况,统计各实验组的斑马鱼死亡数量,确定“实施例5”和“实施例6”对斑马鱼的最大检测浓度(MTC)。
2、评价“实施例5”和“实施例6”抗炎作用
在显微镜下挑选发育健康且阶段一致的受精后3天的(3dpf)转基因中性粒细胞荧光斑马鱼于六孔板中,每孔随机挑选30尾,每孔容量为3mL,卵黄囊注射给予LPS建立斑马鱼炎症模型。分别水溶给予“实施例5”和“实施例6”,浓度均为0.28、0.83和2.5μM;吲哚美辛 的浓度为80μM;同时设置正常对照组(即养鱼用水处理斑马鱼)与模型对照组,置于28℃培养箱中培养3小时后,每组随机取10尾斑马鱼在荧光显微镜下观察、拍照并保存图片;用尼康NIS-Elements D 3.10高级图像处理软件进行图像分析,计算斑马鱼炎症中性粒细胞个数(N),统计学处理结果用mean±SE表示;以斑马鱼炎症中性粒细胞个数的统计学分析结果分别评价“实施例5”和“实施例6”是否对LPS诱发的炎症斑马鱼有显著的抗炎作用。“实施例5”和“实施例6”的抗炎作用计算公式如下:
炎症消退作用(%)=((N(模型对照组)-N(供试品组))/N(模型对照组))*100%
用方差分析和Dunnett’s T-检验进行统计学分析,p<0.05表明具有显著性差异。
实验结果
1、MTC
“实施例5”在DMSO中的最大溶解度为20mM,斑马鱼能够耐受的DMSO最大浓度为1%,因此“实施例5”抗炎作用评价的最大检测浓度为200μM,“实施例5”在200、100、50和10μM浓度时均30尾斑马鱼死亡,死亡率为100%;在5μM浓度时3尾斑马鱼死亡,死亡率为10%;在2.5μM浓度时斑马鱼状态正常,药物未析出,因此“实施例5”抗炎作用评价的最大检测浓度为2.5μM。
“实施例6”在DMSO中的最大溶解度为20mM,斑马鱼能够耐受的DMSO最大浓度为1%,因此“实施例6”抗炎作用评价的最大给药浓度为200μM,“实施例6”在200、100和50μM浓度时斑马鱼均死亡30尾,死亡率为100%;在10和5μM浓度时斑马鱼均死亡4尾,死亡率为13.33%;在2.5μM浓度时斑马鱼状态正常,药物未析出,因此“实施例6”抗炎作用评价的最大检测浓度为2.5μM。
“实施例5”和“实施例6”的最大检测浓度均按照浓度摸索结果进行,即“实施例5”和“实施例6”抗炎作用评价的最大检测浓度均为2.5μM。详见表3。
表3.“实施例5”和“实施例6”在检测浓度下斑马鱼死亡数量统计(n=30)

2、抗炎作用
如表4、图4、图5、图6所示,其中图4虚线区域内为炎症部位中性粒细胞。模型对照组斑马鱼炎症部位中性粒细胞个数(18个)与正常对照组(3个)比较p<0.001,显示LPS诱发转基因中性粒细胞荧光斑马鱼炎症模型建立成功。80μM吲哚美辛组斑马鱼炎症部位中性粒细胞个数(6个)与模型对照组比较p<0.001,其抗炎作用为67%,说明吲哚美辛对炎症斑马鱼有明显抗炎作用。
“实施例5”在浓度为0.28、0.83、2.5μM时,斑马鱼炎症部位中性粒细胞个数分别为12、7和6个,对斑马鱼的抗炎作用分别为33%、61%和67%;与模型对照组(18)比较,0.28、0.83和2.5μM浓度组p均<0.001,提示“化合物3”在浓度为0.28~2.5μM时对炎症斑马鱼具有明显的抗炎作用。
“实施例6”在浓度为0.28、0.83、2.5μM时,斑马鱼炎症部位中性粒细胞个数分别为12、8和7个,对斑马鱼的抗炎作用分别为33%、56%和61%;与模型对照组(18)比较,0.28、0.83和2.5μM浓度组p均<0.001,提示“化合物4”在浓度为0.28~2.5μM时对炎症斑马鱼具有明显的抗炎作用。
表4.各实验组对斑马鱼炎症的影响定量结果(n=10)
实验结论
在本实验浓度条件下,实施例5和实施例6对炎症斑马鱼均有明显的抗炎作用。
实验5评估化合物对大鼠脑缺血再灌注(MCAO)损伤模型的药效学研究
采用神经功能评分和脑梗死面积,评估化合物对小鼠脑缺血再灌注(MCAO)损伤模型的作用。结果显示,测试化合物具有保护小鼠脑缺血再灌注(MCAO)损伤的作用。
实验药品
表5.待测化合物

生理盐水 名称:生理盐水自制。
羟丙基-β-环糊精 名称:羟丙基-β-环糊精;提供单位:萨恩化学技术(上海)有限公司;批次:FG310174;性状:白色固体粉状;数量:30g;贮存条件:常温。
实验方法 实验动物和饲养
实验动物 品系:C57BL/6小鼠;周龄:6-8;性别:雄性;订购动物体重:16-20g;使用动物体重:20-23g;数量:40;实验动物提供商:浙江维通利华实验动物技术有限公司;生产许可证号:SCKX(浙)2019-0001;质量合格证号:No2005130052,No2004280010。
动物饲养
检疫:检疫期7天,常规健康检查由兽医完成,表现异常的动物在实验前剔除。
动物饲养条件:实验动物饲养在动物中心(AAALAC认证单位)的SPF级恒温恒湿的层流清洁房间内,每3只鼠一笼。饲养室温度22±3℃,湿度40-70%,灯光12小时明暗交替。笼具:由聚碳酸酯制成。软制玉米芯高压消毒清洁垫料,每周更换两次。饲料和饮水:清洁级鼠料,购自北京科澳协力饲料有限公司。饮用水经过高压灭菌处理,食物经过钴60射线照射。动物可以自由摄取无菌食物和饮水。
动物编号:每只笼具具有笼具标签,标明动物数量、性别、品系、接收时间、组别以及实验开始时间。动物编号:每只动物在尾部标记单独的动物号。
动物分组及处理,根据动物体重在第1天,将动物随机分为8组,N=5,共8组,每天做一组,每组选3只测脑血流。具体分组及处理见表6。
表6动物分组和药物处理

MCAO模型制作
手术前小鼠禁食一晚,但不禁水。将小鼠置于异氟烷气体麻醉机诱导盒中预麻醉,异氟烷浓度为2.5%。用解剖镊夹钳小鼠后爪无反应后,将其转移至麻醉面罩处,将异氟烷浓度将至1.5%。使用体温维持仪及肛温探头维持小鼠术中体温在37℃附近。剃除小鼠仰卧位颈部毛发,用碘伏和酒精消毒皮肤后,在颈部正中做切口。钝性分离组织,在体视显微镜下曝露颈总动脉(CCA),在其近心端用6-0编织线作结扎。向上分离颈外动脉(ECA)及颈内动脉(ICA),在ECA远心端作永久结扎,ECA远心端作暂时结扎,在颈动脉窦作虚结扎,在此处剪开ECA血管壁,插入线栓(北京西浓科技有限公司,A5-162020),拉紧虚结扎固定线栓。用电凝笔熔断ECA,松开颈外动脉结扎,将线栓插入ICA,直到脑血流量将至基线的10%左右停止。线栓阻塞脑中动脉(MCA)30min,30min后抽出线栓,烧灼血管残端,松开CCA结扎。缝合颈部皮肤,小鼠置于重症监护笼中,维持小鼠术后体温在37℃,直到取材前。
脑血流的测量:在脑立体定位仪下固定小鼠头部,剃除小鼠头部毛发,作正中切口。去除颅骨上的骨膜,将激光多普勒血流仪光纤用胶水固定在坐标为Bregma AP 1.0mm,ML5.0mm处,记录脑中动脉术中血流变化。造模成功标准为脑血流量下降到基线的80%-90%。
药物配制
溶媒:生理盐水
溶媒:20%羟丙基-β-环糊精
配制PK样:首先根据每组鼠的总重量,以及给药剂量,算得一天至少所需药量m2。称取稍多于m2的量的P2于一瓶中,并按给药体积10mL/kg,算得溶媒体积v2。向瓶中加入20%羟丙基-β-环糊精溶液(v2),涡旋一分钟,超声15分钟,再涡旋一分钟,直至溶清。
溶媒:20%羟丙基-β-环糊精
配制PK样:首先根据每组鼠的总重量,以及给药剂量,算得一天至少所需药量m3。称取稍多于m3的量的P3于一研钵中,并按给药体积10mL/kg,算得溶媒体积v3,并用注射器量取v2的溶媒。向研钵中加入2-3滴溶媒,研磨5分钟,待溶剂研干后,再加入2-3滴溶媒,再研磨5分钟,重复三次。然后,少量多次将研钵中的化合物用溶媒洗干净并用干净的玻璃滴管加入到一合适的样品瓶中,超声15分钟,再涡旋五分钟,直至均匀,无较大颗粒存在。
检测指标及方法
体重:记录小鼠手术前的当天及术后24h体重。
神经功能评分:小鼠手术后2h、24h进行Longa行为学评分。
Longa行为学评分标准:
0分:无神经损伤症状;
1分:不能完全伸展对侧前爪;
2分:向对侧转圈;
3分:向对侧倾倒;
4分:不能自发行走,意识丧失。
前肢抓力测试
小鼠手术前24h和术后24h分别进行前肢抓力测试,将小鼠置于抓力计上,小鼠主动拉抓力感应杆,记录其抓力峰值,重复测量三次取其平均值。
梗死面积
手术后24h用异氟烷将小鼠麻醉后,断头处死。剥离全脑,用生理盐水涮洗2次,将全脑置于冠状面脑模具下,去除前1mm嗅球和后4mm小脑,将大脑切成2mm一片,共4片。用1%TTC溶液在37°避光处孵育脑片染色20min,再转移至4%PFA4℃避光保存48小时后拍照。用软件ImageJ测量脑梗死体积及水肿体积。
脑水肿体积=(损伤侧半脑体积-对侧半脑体积)/对侧半脑体积*100%校正的脑梗死体积=(对侧半脑体积-(损伤侧半脑体积-白色梗死区体积))/对侧半脑体积*100%
统计
各组数据采用Graghpad Prism 7.0进行数据统计,实验结果以“均值±标准差”表示。统计方法使用单因素方差分析,比较各组之间有无统计学差异,P<0.05具有统计学差异。
实验结果
如图7所示,在MCAO手术中,各组(n=3)脑血流均降至基线的10%左右,说明模型成功可以进行药效评价。
如图8所示,在手术后各组体重均有下降,但经MCAO手术各组小鼠的体重无显著性差异。
如图9所示,手术后各组小鼠前肢抓力均明显下降,其中术后假手术组与MCAO模型组具有显著性差异(P<0.001),MCAO模型组与阳性对照药依达拉奉组有显著性差异(P<0.001),MCAO模型组与3号给药组有显著性差异(P<0.001)。
如图10所示,手术后2h MCAO模型组的神经功能缺损评分与2号给药组及3号给药组分别有显著性差异(P<0.05);手术后24h MCAO模型组的神经功能缺损评分与阳性对照药依达拉奉组及3号给药组分别有显著性差异(P<0.05)。
如图11所示,TTC染色结果显示MCAO模型组脑梗死体积分别与阳性对照药依达拉奉组(P<0.001),2号(P<0.001),3号(P<0.001),4号(P<0.01)以及5号药(P<0.01)有显著性差异。
如图12所示,TTC染色结果显示MCAO模型组脑水肿体积分别与阳性对照药依达拉奉组(P<0.01),2号(P<0.001),3号(P<0.001),4号(P<0.001),5号药(P<0.05)以及6号药(P<0.05)有显著性差异。
实验6评估实施例6在LPS诱导的小鼠抑郁样模型的作用
采用LPS诱导小鼠产生外周及中枢炎症进而使小鼠产生抑郁样症状,检测用药后小鼠抑郁相关行为学指标变化。结果显示,实施例6(ND)具有抗小鼠抑郁作用。
表7待测化合物
溶媒 羧甲基纤维素钠(NC);名称:羧甲基纤维素钠;提供单位:迈瑞尔;批次:69881020;性状:白色固体粉状;数量:200g;贮存条件:常温
外周及中枢炎症诱导剂(LPS)名称:脂多糖;生产单位:Sigma;批次:L2880-100MG;性状:白色粉末;纯度:>97%;规格:100mg;贮存条件:2-8℃,避光
试验方法 实验动物和饲养管理
实验动物品系:C57BL/6J小鼠;周龄:6-8周;性别:雄性;动物体重:18-20g;数量:24;实验动物提供商:广东省医学实验动物中心
动物饲养
检疫:检疫期5天,表现异常的动物在实验前剔除。
动物饲养条件,实验动物饲养在北京大学深圳研究生院动物中心清洁级恒温恒湿的层流清洁房间内,每笼4-5只。饲养室温度22±3℃,湿度40-70%,灯光12小时明暗交替。饲料和饮水:清洁级鼠料,购自北京科奥协力饲料有限公司。饮用水经过高压灭菌处理。动物可以自由摄取无菌食物和饮水。
动物编号,每只笼具具有笼具标签,标明动物数量、性别、品系、接收时间、组别以及实验开始时间。动物编号:每只动物在尾部标记单独的动物号。
动物分组及处理,将动物随机分为3组,具体分组及处理见表8。
表8动物分组和药物处理
LPS诱导的抑郁样模型建立
造模方法:第1、2、3天注射LPS(2mg/kg,i.p.,QD)诱导小鼠外周及中枢炎症。ND药物测试组于注射LPS前2h给药进行预保护(ND 30mg/kg,p.o.,QD),于实验D4进行强迫游泳、悬尾及糖水偏好实验。
供试药和模型诱导剂配制
溶媒(0.5%CMC-Na溶液):取0.5g CMC-Na白色固体粉末,加入100毫升双蒸水中,涡旋直至溶清。配制好的溶液应置于4度冰箱,密封保存。该溶液可保存一月,但如若发现有发霉现象,禁止用于实验,需重新配制。
试验期间待测化合物和模型诱导剂现用现配,具体配置方法如表9:
表9溶媒及供试药配制
检测指标及方法
一般状态观察,观察记录动物的生活状态。
强迫游泳实验(Forced swim test,FST)
将小鼠分别放入一个透明玻璃圆筒(直径:23厘米;高度:31厘米),其中装有15厘米的水,温度保持在24±1摄氏度。FST持续6分钟,期间用高清摄像头拍摄,专业测试软件计算小鼠在测试过程中的不动时间,结束后小鼠立即放回笼子,并注意保温。
糖水偏好实验(Sucrose preference test,SPT)
在测试前对小鼠进行训练,即每笼放入2瓶1%(W/V)蔗糖溶液,24h后将其中一瓶换为纯水。适应结束后禁食禁水10-24h后进行测试。每笼放置2个预先称重的水瓶,一瓶为1%(W/V)蔗糖溶液,一瓶为纯水,12h后取两个水瓶再次称重,记录各小鼠糖水消耗和纯水消耗。
糖水偏好指数%=糖水消耗量/(糖水消耗量+纯水消耗量)×100%。
悬尾实验(Tail suspension test,TST)
经环境适应后,将小鼠尾巴粘贴于悬挂杆上,小鼠头部距地约20-25cm,持续约6min。高清摄像机拍摄录像,并用行为学分析软件识别统计小鼠不动时间。实验结束后将小鼠放回笼子。
统计
实验结果以“均值±标准差”表示。各组数据采用One-way ANOVA比较各组之间有无统计学差异。
实验结果
FST
如图13所示,各组小鼠FST不动时间结果表明,实施例6(ND)累计不动时间显著低于LPS组。**,P<0.01;***,P<0.001,
SPT
如图14所示,各组小鼠SPT中糖水偏好率结果表明,实施例6(ND)与LPS组相比没有显著提高。*,P<0.05;***,P<0.001
TST
如图15所示,各组小鼠TST不动时间结果表明,实施例6(ND)累计不动时间显著低于LPS组。*,P<0.05;***,P<0.001
结论:化合物实施例6(ND)在FST、TST实验中表现出较好的抗抑郁效果。
实验7.评估化合物实施例20A在LPS诱导的小鼠外周及中枢炎症模型上的作用
采用LPS诱导小鼠产生外周及中枢炎症,检测用药后小鼠血清及海马组织内炎症及氧化应激水平的变化。结果显示,实施例20A(ND)具有明显抗炎作用。
供试品
表10待测化合物
溶媒
羧甲基纤维素钠(NC),名称:羧甲基纤维素钠;提供单位:迈瑞尔;批次:69881020;性状:白色固体粉状;数量:200g;贮存条件:常温。
外周及中枢炎症诱导剂(LPS),名称:脂多糖;生产单位:Sigma;批次:L2880-100MG;性状:白色粉末;纯度:>97%;规格:100mg;贮存条件:2-8℃,避光。
试验方法
实验动物和饲养管理
实验动物品系:C57BL/6J小鼠;周龄:6-8周;性别:雄性;动物体重:18-20g;数量:24。实验动物提供商:广东省医学实验动物中心
动物饲养
检疫,检疫期5天,表现异常的动物在实验前剔除。
动物饲养条件,实验动物饲养在北京大学深圳研究生院动物中心清洁级恒温恒湿的层流清洁房间内,每笼4-5只。饲养室温度22±3℃,湿度40-70%,灯光12小时明暗交替。饲料和饮水:清洁级鼠料,购自北京科奥协力饲料有限公司。饮用水经过高压灭菌处理。动物可以自由摄取无菌食物和饮水。
动物编号,每只笼具具有笼具标签,标明动物数量、性别、品系、接收时间、组别以及实验开始时间。动物编号:每只动物在尾部标记单独的动物号。
动物分组及处理
将动物随机分为3组,具体分组及处理见表11。
表11动物分组和药物处理
LPS炎症模型建立
造模方法:第1、2、3天注射LPS(2mg/kg,i.p.,QD)诱导小鼠外周及中枢炎症。ND药物测试组于注射LPS前2h给药进行预保护(ND 30mg/kg,p.o.,QD),实验结束后收集小鼠血浆及脑组织进行炎症因子和过氧化因子等指标。
供试药和模型诱导剂配制
溶媒(0.5%CMC-Na溶液):取0.5g CMC-Na白色固体粉末,加入100毫升双蒸水中,涡旋直至溶清。配制好的溶液应置于4度冰箱,密封保存。该溶液可保存一月,但如若发现有发霉现象,禁止用于实验,需重新配制。
试验期间待测化合物和模型诱导剂现用现配,具体配置方法如表12:
表12:溶媒及供试药配制

检测指标及方法
一般状态观察,观察记录动物的生活状态。
动物取材
血清:采用1%戊巴比妥钠对小鼠进行麻醉后摘眼球取血于EP管中,4℃静置1h后低温离心,取上清,至于液氮中速冻。
海马组织:采用1%戊巴比妥钠对小鼠进行麻醉后用冷PBS进行心脏灌注,取脑,冰上取组织于EP管液氮速冻。
氧化应激相关指标测定
取冻存血浆,采用活性氧检测试剂盒、过氧化氢检测试剂盒、一氧化氮检测试剂盒进行各项指标检测,具体操作流程同各试剂盒说明书。
取冻存海马组织,加入含蛋白酶抑制剂的RIPA组织裂解液,充分匀浆裂解,低温离心后取上清液,采用BCA蛋白定量试剂盒进行蛋白定量。调整各组织样品至统一蛋白浓度,采用活性氧检测试剂盒、过氧化氢检测试剂盒、一氧化氮检测试剂盒进行各项指标检测,具体操作流程同各试剂盒说明书。
细胞因子检测
取冻存血浆,采用IL-1β、IL-6、IL-10试剂盒进行各项指标检测,具体操作流程同各试剂盒说明书。
取上述海马组织上清液,采用IL-1β、IL-6、IL-10试剂盒进行各项指标检测,具体操作流程同各试剂盒说明书。
统计
实验结果以“均值±标准差”表示。各组数据采用One-way ANOVA比较各组之间有无统计学差异。
实验结果
氧化应激相关指标
如图16~23所示,各组小鼠血清及海马组织氧化应激重要指标检测结果表明,实施例20A(ND)血清中的ROS水平、H2O2浓度、NO浓度相对于LPS组都有明显改善,表明实施例20A(ND)有很好的抗氧化作用。*,P<0.05;**,P<0.01。
炎症因子指标
如图24~29所示,各组小鼠血清及海马组织炎症相关重要指标检测结果表明,实施例20A只影响了血清中的IL-1β、海马中的IL-10,表明实施例20A(ND)有抗炎作用。*,P<0.05;**,P<0.01。
实验8小分子化合物对线粒体呼吸链超级复合物的结合强度检测
实验与分析方法:在5%CO2的条件下培养HEK293细胞并从中纯化出线粒体呼吸链超级复合物I1III2IV1。将呼吸链超级复合物通过化学共价偶联到金属芯片表面后,再用不同浓度的小分子化合物溶液流过金属芯片,通过Biacore 8K plus分析仪采用SPR技术监测金属表面反射系数的改变,继而绘制出小分子结合的相应响应曲线图。通过Biacore 8K plus仪器内置拟合软件计算出小分子与人源线粒体呼吸链超级复合物I1III2IV1的结合强度(KD值)。
化合物处理:将图示小分子用ddH2O溶解配置成10mM储液。然后用10mM的HEPES(pH=7.4)溶液将实施例13B储液梯度稀释为1.5625μM、0.78125μM、0.390625μM和0.1953125μM。同样的,用10mM的HEPES(pH=7.4)溶液将实施例1A储液梯度稀释为6.25μM、3.125μM、1.5625μM、0.78125μM、0.390625μM和0.1953125μM。测定时,取200μL各浓度小分子溶液流经金属芯片表面。
结果如图30a至32b所示,小分子与呼吸链复合物I的结合强度都达到了很高的水平。
实验9.小分子化合物促进SMP(亚线粒体颗粒)催化活性检测
实验与分析方法:在5%CO2的条件下培养HEK293细胞并从中纯化出线粒体,将线粒体超声处理得到的SMP(亚线粒体颗粒)。将SMP与不同浓度的小分子化合物或对照药(Rotenone,2μM)孵育10分钟后,加入500μM的NADH。通过Enspire多模式微孔酶标仪检测NADH吸光度,绘制出SMP催化导致NADH浓度降低的酶活曲线,线性拟合并计算出SMP的最大反应速率。在SMP的正常NADH催化体系中加入1mM的FeCN,可以导致从NADH到辅酶Q的电子通路短路。在FeCN和Rotenone存在的体系下,同样测定NADH浓度降低曲线,线性拟合并计算SMP黄素位点的最大反应速率。
化合物处理:将图示小分子用ddH2O溶解配置成10mM储液,然后用10mM的Tris缓冲溶液将小分子梯度稀释到0.075、0.1、0.133、0.178、0.237、0.316、0.422、0.563、0.75、1μM来进行SMP催化实验。
结果如图33所示,在催化实验体系中,对照组具有催化活性,Rotenone完全阻断了NADH的脱氢反应,反应体系有效。1μM以内的图示小分子对SMP的活性无显著的影响。而高浓度的图示小分子则对SMP的活性有较大的影响。
实验10.小分子化合物促进HPAEC细胞线粒体应激检测。
实验与分析方法:从ATCC处购买HPAEC细胞(人源肺动脉内皮细胞),并在5%CO2培养条件下传代4次后转移到Seahorse专用细胞板中,每孔约2万个细胞。待细胞贴壁后,用0.5、1μM的实施例13B与HPAEC细胞孵育48小时后使用Seahorse XFe96分析仪检测每孔细胞耗氧速率(OCR),并绘制OCR曲线。测定结束后,通过BCA测定每个细胞孔中的总蛋白含量并对OCR曲线进行校正。
结果如图34所示,在静息状态时(时间:0-20分钟),5种化合物对细胞耗氧几乎没有影响。加入复合物V的抑制剂oligomycin后,质子梯度不被消耗,高质子梯度会抑制复合物I的活性,导致耗氧速率降低,但不同小分子和对照组的降低幅度几乎没有差别。加入解偶联剂FCCP,质子梯度骤降,耗氧大幅增加,所有小分子都提高了耗氧速率的增幅,这反映了这些小分子可以提高复合物I对质子梯度骤降进行反应的敏感度,促使复合物I更强地提高电子传递速率。最后加入Rotenone(线粒体电子传递链复合物I抑制剂)和Antimycin(线粒体电子传递链复合物III抑制剂),完全阻断电子传递,耗氧速率降低至最低值,小分子对电子传递的阻断无影响。
实验11.小分子化合物保护HPAEC细胞免受新冠病毒Spike诱导ROS产生的检测。
实验与分析方法:从ATCC处购买HPAEC细胞(人源肺动脉内皮细胞),并在5%CO2培养条件下传代4次后转移到MatTek细胞成像皿中,每槽约5万细胞。HPAEC细胞与Spike(新冠病毒表面刺突蛋白,8μg/mL)孵育培养24小时或与实施例13B(0.25、0.5、1μM)预孵育6小时后与Spike(8μg/mL)孵育培养24小时。随后,使用MitoSox Red、PKMDR、Mitotracker Green线粒体荧光探针进行线粒体染色,并通过Leica受激发射损耗超高分辨率共聚焦荧光显微镜(STED)拍摄细胞荧光共聚焦照片。后续用ImageJ测量不同组内细胞荧光强度,利用所得到的数据计算标准差和标准误差,做T-test分析计算P值,确定是否有显著性差异。
结果:HPAEC细胞加入Spike之后,线粒体ROS相对于对照组明显升高(红色荧光更亮)。如图35a至35b所示,图示小分子保护线粒体之后加入Spike,线粒体ROS有明显的回落;利用PKMDR/MTG的值作为衡量单位质量线粒体跨膜电势差的指标,图示小分子对线粒体跨膜电势差具有保护作用。
实验12.小分子化合物改善APOE小鼠动脉粥样硬化的检测
实验与分析方法:购买6周龄雄性APOE-/-突变B6J小鼠和WT的B6J小鼠后先在饲养房间内适应2周。当小鼠8周龄时除了Vehicle组和WT组之外,其余组开始给高脂饲料(Research Diets,D12492)。当小鼠9周龄时,Control组给10%环糊精溶液,其余给药组 开始分组经口灌胃不同浓度小分子化合物,灌胃频率每2天一次。当给药满1月时,对APOE小鼠进行眼眶静脉从采血,采血体积约500mL,离心后吸取上清血浆并送检测定血浆中的总胆固醇(CHOL)、高密度脂蛋白(HDL)和低密度脂蛋白(LDL)水平。当高脂饲喂满16周后,处死APOE小鼠并解剖取材主动脉弓。小鼠主动脉弓在用4%多聚甲醛固定后再使用油红对主动脉弓内部动脉斑块进行染色,并通过蔡司体式显微镜对染色后动脉弓进行成像。拍摄的照片用ImageJ分析动脉弓纵剖面整体面积和橙红色斑块的面积,计算获得斑块面积占比并对数据做T-test分析计算P值,确定是否有显著性差异。
化合物处理:将图示小分子(图36a至36d)解到10%环糊精溶液中,达到终浓度12mg/mL。然后用10%环糊精溶液梯度稀释到6、3、1.5mg/mL。
结果如图36a至36d所示:Control组的APOE小鼠在高脂饲喂后血清中总胆固醇水平接近800mg/dL,而WT基因型的小鼠血清总胆固醇仅约100mg/dL。给图示小分子后,APOE小鼠的总胆固醇水平均有降低。同时,图示小分子基本不影响APOE小鼠的高密度脂蛋白,而低密度脂蛋白呈现的趋势和总胆固醇几乎一致,这说明图示小分子主要是降低了不好的LDL而能维持住有益的HDL。对APOE动脉弓的油红染色能明显看出给药组的橙红色斑块区域比Control组少很多,这从统计上也有显著性差异。以上结果说明图示小分子能降低血清LDL水平从而抑制动脉粥样硬化的发生。
实验13.AD大鼠水迷宫
实验与分析方法:使用CRISPR Cas9技术将人源APP突变蛋白导入SD大鼠胚胎,得到转基因AD大鼠模型。该品系的大鼠在2月龄开始就有淀粉样蛋白在大脑里沉积,5月龄时开始表现出一定的行为学障碍。为了验证治疗效果,从7月龄开始给药,给到13月龄,做水迷宫实验验证对空间记忆的改善。水迷宫实验的过程分为三个阶段:第一阶段1天,为适应阶段,在1.8m直径的大水池中有一个15cm直径的逃脱平台,水池壁上东西南北四个方位有圆圈、三角、方框、叉四个记号作为空间位置的提示。将大鼠依次沿池壁放入透明的水中,水面在逃脱平台之下1cm。大鼠将很容易找到逃脱平台,爬上平台之后被救出水迷宫,从而学会水迷宫的的基本规则。第二阶段4天,为训练阶段。水池设置不变,将水面抬高到逃脱平台以上1cm,并倒入墨水将水染成黑色,使逃脱平台不可见。每天将大鼠从东南西北四个方位放入水池中,成功爬上逃脱平台之后救出。如90s内没有找到平台,则人工引导大鼠爬上平台,在平台上站立15s后救出。第三阶段1天,为考试阶段。将逃脱平台取出,大鼠依旧沿池壁放入一次。空间记忆强的大鼠将在原有逃脱平台的位置反复穿梭。记录第一次到达平台的潜伏期、平台穿梭次数、跟平台的平均距离、目标象限的停留时间等指标。
结果:总共有三组小鼠,WT组13只,模型组(AD组)7只,给药组(实施例13B组)11只,第一次到达平台的潜伏期(如图37a)、平台穿梭次数(如图37b)、跟平台的平均距离(如图37c)、目标象限的停留时间(如图37d)等指标上,给药组的大鼠相较于模型组,行为学表现都有所改善。图37e至图37g显示各组大鼠在水池中的运动轨迹热图。
实验14.AD大鼠T迷宫
实验与分析方法:使用CRISPR Cas9技术将人源APP突变蛋白导入SD大鼠胚胎,得到转基因AD大鼠模型。该品系的大鼠在2月龄开始就有淀粉样蛋白在大脑里沉积,5月龄时开始表现出一定的行为学障碍。为了验证治疗效果,从7月龄开始给药,给到15月龄,做T迷宫实验验证对工作记忆的改善。T迷宫有四个阶段:I.适应阶段,两臂均放有食物,两臂阀门均打开。每只大鼠训练2次。当所有大鼠两次都在5min内吃完两侧食物之后进入下一阶段。II.迫选训练阶段,先关闭一侧阀门,让大鼠吃完对侧食物。再关闭对侧阀门,让大鼠吃完另一侧食物。此为一次训练。每只动物每天训练4次,持续3天。III.自选训练阶段,先关半一侧阀门,让大鼠吃完对侧食物。再打开所有阀门,如果大鼠去到另一侧,可以吃完在那儿的食物。如果大鼠去到同侧,则没有食物,并被关闭在这一侧30s。此为一次训练。每只动物每天训练4次,直到正确率连续2天超75%进入下一阶段。IV.延时自选测试阶段,与第 III阶段操作基本相同,区别在于迫选结束开始自选之前,加入间隔时间。间隔时间设置为1.5min,3min,10min。每个时间测试4次。
结果:总共有三组小鼠,WT组3只,模型组(AD组)2只,给药组(实施例13B组)6只。如图38所示,在第1.5min和3min时,模型组和给药组没有明显的区别,但是在第10min时,模型组的工作记忆明显减弱,而给药组的工作记忆还持续存在。
实验15.AD小鼠筑巢行为
实验与分析方法:本实验使用APP/P实施例13B导入的转基因小鼠,背景鼠是C57BL/6J。该模型鼠的表型为3月龄出现认知行为学变化,5月龄出现老年斑,12月龄有大量老年斑形成。AD病人的一个症状是生活不能自理,而筑巢行为反应的是小鼠的生活自理水平。实验开始时,将10张长方形的纸片按顺序整齐摆放入鼠笼内。正常小鼠会将纸片撕碎并搭建鼠窝,而AD症状严重的小鼠则不会撕碎纸片搭窝。依据纸片的撕碎程度和搭窝的完整性进行评分,评分越高的小鼠筑巢行为越好,反应生活自理水平越高。
结果:本实验从2月龄开始给药,在给药3月后和给药6月后做了筑巢行为评分测试。测试了四种小分子,实施例23,实施例24,实施例25和实施例26。结果如图39a和39b显示,模型组相较于WT组筑巢行为有明显的减弱,而实施例25对筑巢行为的改善最明显。
实验16.AD小鼠水迷宫
实验与分析方法:本实验使用APP/P实施例13B导入的转基因小鼠,背景鼠是C57BL/6J。该模型鼠的表型为3月龄出现认知行为学变化,5月龄出现老年斑,12月龄有大量老年斑形成。水迷宫实验的过程分为三个阶段:第一阶段1天,为适应阶段,在1.2m直径的大水池中有一个8cm直径的逃脱平台,水池壁上东西南北四个方位有圆圈、三角、方框、叉四个记号作为空间位置的提示。将大鼠依次沿池壁放入透明的水中,水面在逃脱平台之下1cm。小鼠将很容易找到逃脱平台,爬上平台之后被救出水迷宫,从而学会水迷宫的的基本规则。第二阶段4天,为训练阶段。水池设置不变,将水面抬高到逃脱平台以上1cm,并倒入墨水将水染成黑色,使逃脱平台不可见。每天将小鼠从东南西北四个方位放入水池中,成功爬上逃脱平台之后救出。如90s内没有找到平台,则人工引导大鼠爬上平台,在平台上站立15s后救出。第三阶段1天,为考试阶段。将逃脱平台取出,小鼠依旧沿池壁放入一次。空间记忆强的大鼠将在原有逃脱平台的位置反复穿梭。记录平台穿梭次数、目标象限的停留时间等指标。
结果:本实验从2月龄开始给药,在给药6月后做了水迷宫评价空间记忆。测试了四种小分子,实施例23,实施例24,实施例25和实施例26。结果如图40a和40b显示,模型组相较于WT组筑空间记忆能力有明显的减弱,而从平台穿梭次数、目标象限停留时间等指标上看实施例25对空间记忆能力的改善最明显。
实验17.AD小鼠明暗箱
实验与分析方法:本实验使用APP/P实施例13B导入的转基因小鼠,背景鼠是C57BL/6J。该模型鼠的表型为3月龄出现认知行为学变化,5月龄出现老年斑,12月龄有大量老年斑形成。明暗箱实验的原理是:小鼠有趋暗的天性,将小鼠放入明箱以后,小鼠会自发地进入相连通的暗箱里。但是在本实验中,暗箱装有电刺激的机关,小鼠进入暗箱后会被电击。于是,正常小鼠在被电击数次后就不再进入暗箱,而AD小鼠则不记得进入暗箱后会被电击,将会继续进入暗箱。所以,将小鼠放入明箱后,小鼠自发进入暗箱前的潜伏期,则反映了小鼠对电击的记忆强度,越晚进入暗箱则对电击的记忆越强。
结果:本实验从2月龄开始给药,在给药6月后做了水迷宫评价空间记忆。测试了四种小分子,实施例23,实施例24,实施例25和实施例26。结果如图41显示,模型组相较于WT组对电击的记忆有明显的减弱,而实施例23和实施例25两种小分子则可以有效地增强模型小鼠对电击的记忆。
实验18.TDP43A315T小鼠生存期
实验与分析方法:本实验使用TDP43A315T转基因小鼠,该品系的小鼠会模拟ALS(渐冻 症)的发病过程。该品系小鼠一般在90天的时候开始死亡,在120天左右达到死亡中位数。我们的实验分为两组,模型组和给药组。模型组不给药,给药组从第60天开始给药,测试小分子实施例13B,剂量为40mpk。每组小鼠20只,本实验记录每一只小鼠的死亡日期以及体重,绘制体重曲线和生存曲线。
结果:体重是反应ALS病程的一个重要指标。如图42a所示,可以看到,模型组小鼠的体重在13周就开始大幅下降,而给药组小鼠则从16周才开始大幅下降。如图42b所示的生存曲线,可以看到,模型组小鼠从90天开始出现大量死亡,死亡中位数为120天左右,140天左右全部死亡。而给药组100天以后才开始死亡,120天以后开始大量死亡,死亡中位数在135天,150天才全部死亡。结果显示,小分子实施例13B可以有效地延长TDP43A315T转基因小鼠的生存期,说明该小分子可能可以有效地延长ALS病人的生存期。
实验19.SODG93A小鼠步态分析
实验与分析方法:本实验使用SODG93A转基因小鼠,该品系的小鼠会模拟ALS(渐冻症)的发病过程,随着周龄的增长会有明显的运动功能障碍发生。步态分析是测试运动功能障碍的一种行为学分析方法。有运动功能障碍的小鼠步幅会明显减小。我们的实验分为两组,模型组和给药组。模型组不给药,给药组从第60天开始给药,测试小分子实施例13B,剂量为40mpk。每组小鼠10只,在第19周进行,测试每只小鼠的步幅。
结果:图43显示,给药组小鼠的步幅要远大于模型组小鼠,说明小分子实施例13B显著改善了SODG93A小鼠的运动能力。
实验20.SODG93A小鼠旷场
实验与分析方法:本实验使用SODG93A转基因小鼠,该品系的小鼠会模拟ALS(渐冻症)的发病过程,随着周龄的增长会有明显的运动功能障碍发生。在旷场中计算平均移动速度是测试运动功能障碍的一种行为学分析方法。有运动功能障碍的小鼠平均移动速度会明显减小。我们的实验分为三两组,WT组,模型组和给药组。WT组为正常小鼠,模型组不给药,给药组从第60天开始给药,测试小分子实施例13B,剂量为40mpk。每组小鼠10只,在第14周进行,每周按时测试每只小鼠在旷场中的平均运动速度,直到第19周。
结果:图44显示,在测试的6周内,给药组小鼠的平均运动速率要远大于模型组小鼠,说明小分子实施例13B显著改善了SODG93A小鼠的运动能力。
实验21.DSS小鼠肠炎模型
实验与分析方法:本实验目的是考察受试物(实施例13B,实施例33,实施例34)经口灌胃多次给药,对3.0%葡聚糖硫酸钠(DSS)诱导的小鼠溃疡性结肠炎是否有治疗作用。将健康状态良好的60只小鼠根据体重平均分为6组,每组10只:正常对照组、模型组、阳性药组、不同种类受试物(实施例13B,实施例33,实施例34,均为80mpk)组。除正常对照组外的其它小鼠给予3.0%DSS溶液自由饮水造模,连续造模8天;正常对照组给予高压灭菌过滤水自由饮水。造模当天开始给药,受试物根据设计剂量经口灌胃给药,一天一次,连续9天。给药期间,每日对小鼠体重进行监测,观察小鼠的粪便形状、有否出血等。末次给药次日,CO2深度麻醉后,心脏采血,收集血浆,置于-80℃冻存;随后剖开腹腔,摘取肝脏,置于-80℃冻存;最后摘取整个结肠组织,测量结肠长度,拍照,沿肠系膜侧剖开后用生理盐水清洗结肠内容物并称重。取部分病变部位组织用10%福尔马林固定,进行HE染色组织病理学观察,其余结肠组织-80℃冻存。
结果:(1)如图45a所示,对DSS结肠炎小鼠一般状况的影响:3.0%DSS造模4天后,造模动物陆续出现稀便、血便、体重下降等异常情况,之后逐渐加剧;模型组小鼠伴有饮食减少、疲倦、消瘦、竖毛、毛发无光泽的症状。720mg/kg阳性药SASP和受试物(实施例13B,实施例33,实施例34)均有一定改善作用,但至实验终点仍有体重下降及稀便、血便等异常情况。(2)如图45b所示,对DSS结肠炎小鼠结肠重量及长度的影响:自由饮用3.0%DSS溶液后小鼠结肠出现萎缩现象,表现为结肠长度缩短和重量减轻;阳性药和受试物均可抑制小 鼠结肠萎缩;受试物(实施例13B,实施例33,实施例34)均可增加DSS小鼠结肠重量和长度,抑制小鼠结肠萎缩。
实验22.TNBS大鼠肠炎模型
实验与分析方法:本实验目的是研究多次经口灌胃给予受试物(实施例13B,实施例36,实施例37,均为80mpk)对2,4,6-三硝基苯磺酸(TNBS)诱导的大鼠溃疡性结肠炎是否有治疗作用。除正常对照组外的所有大鼠禁食(不禁水)24h,以异氟烷麻醉后,大鼠采用乳胶软管经直肠灌入0.5mL/只的TNBS乙醇溶液(18mg TNBS/只),软管进入直肠的长度约为8cm,抽出软管后继续保持麻醉状态并使大鼠保持倾斜状态15min;正常对照组大鼠不进行直肠插管刺激。待动物清醒后随机分组,随即开始给药(记为D1),空白组和模型组给予等量溶媒,经口灌胃给药,一次/日,连续7天(D1-D7);每日观察动物状态,粪便情况(有否稀便、血便),并监测体重。末次给药后次日(D8),CO2深度麻醉,处死后解剖动物,摘取整个结肠组织,沿肠系膜侧剖开后用生理盐水清洗结肠内容物,称重并测量结肠长度,Image J软件测量溃疡面,观察结肠大体情况并拍照。取病变部位组织用福尔马林固定,进行HE染色组织病理学观察。
结果:(1)如图46a所示,对TNBS致溃疡性肠炎大鼠一般情况的影响:TNBS造模次日大鼠有稀便发生;造模大鼠伴有饮食减少、疲倦、消瘦、竖毛、毛发无光泽的症状。部分大鼠给药后期粪便有所改善,但仍有部分大鼠稀便状况维持到实验终止,个别大鼠实验后期出现明显腹胀情况;(2)如图46b所示,对TNBS致结肠炎大鼠结肠重量及长度的影响:综合大鼠单位长度的结肠重量(结肠重量/结肠长度)和结肠溃疡面积的结果可知,受试物(实施例13B,实施例36,实施例37)均可减小大鼠的单位长度结肠重量和结肠溃疡面积,其中实施例13B作用更显著。
实验23.DB小鼠血糖降低
实验与分析方法:瘦素受体基因(Obese Gene Receptor,OB-R)又称为糖尿病基因(Diabetes Gene,db),瘦素受体(leptin receptor,Lepr)与肥胖、高血压、糖尿病、脂质代谢紊乱等之间有密切关系。该疾病病程受基因背景影响很大。外用胰岛素无法控制血糖和肝脏葡萄糖异生水平的升高。利用基因编辑技术和胚胎注射技术构建Lepr基因突变小鼠。通过对该品系纯合子的血糖监测发现,其血糖水平与野生对照相比呈显著性差异,可用于II型糖尿病研究。该品系小鼠8周龄开始血糖飙升,达到30mmol/L,而正常的背景鼠血糖仅为6mmol/L左右。我们从8周龄开始给药,测试小分子为实施例28,实施例29和实施例30,课题均为80mpk,每天一次灌胃给药。每组小鼠20只,从8周龄开始,每周监测血糖,直到20周龄处死。
结果:结果如图47显示,实施例28和实施例29都有一定的降血糖效果,但是实施例30的降血糖效果最好,可以达到正常背景鼠的水平。
实验24.DIO小鼠体重下降,体脂率下降
实验与分析方法:DIO模型为高脂饲喂导致的肥胖模型。本实验使用C57BL/6J和C57BL/6N两个品系的小鼠,使用含有60%脂肪的高脂饲料。在小鼠7周龄时换高脂饲料进行造模,持续高脂饲喂5个月,使小鼠体重达到50g以上,开始给药,并每周记录体重,每两周使用Echo MRI小动物体成分分析仪进行体脂率的测定。我们测试了5种小分子的减肥效果,实施例33,实施例34,实施例35,实施例36,实施例37,剂量为80mpk,2天一次灌胃给药。
结果:如图48a至48d所示,实施例34、实施例35的减肥效果最佳,可以将肥胖模型鼠的体重从50g以上减到35g左右的正常小鼠水平。另外实施例33、实施例36、实施例37也有减肥效果,可以减到40g左右。从体脂率来看,所有的小分子都能有效地燃烧小鼠体内的脂肪、降低体脂率,依然是实施例34、实施例35的燃脂效果最佳,实施例33、实施例36、实施例37次之。
实验25.NOD-Scid免疫缺陷小鼠接种血液瘤细胞Raji-Luc
实验与分析方法:本实验目标为测试6种小分子对血液瘤的体内杀伤效果。本实验使用8周龄的雌性NOD-Scid免疫缺陷小鼠作为Raji-Luc细胞的接种鼠,总共80只,接种后第9天进行活体荧光成像,依据荧光强度,选择中段荧光强度值最集中的60只进行后续实验。第一次活体成像后开始给药,剂为100mpk每天一次灌胃给药,测试的小分子为实施例25A、实施例25B、实施例28、实施例38、实施例39、实施例40,每组10只。开始给药后每周两次进行活体成像,以观察小鼠全血肿瘤的发展情况。
结果:如图49所示,可以看到,control组荧光强度非常高,而给药组的荧光强度都有明显的下降。其中实施例28和实施例38两组毒性过高,每21天全部死完,没有荧光值。另外的实施例25A、实施例25B、实施例39、实施例40都有比较好的杀肿瘤效果。
实验26.小分子化合物细胞增殖检测
实验与分析方法:将细胞接种到带盖的96孔细胞培养板,测试化合物最高浓度为10μM,3倍稀释到一系列测试浓度,细胞在37℃、5%CO2中孵育72小时。DMSO浓度为0.5%。从每个孔取出100ul培养基。每孔加入100ul试剂(Celltitute Glo分析试剂盒),并在平板振荡器上摇动平板(避光)2分钟。在室温下孵育培养板(避光)30分钟。通过多板读板仪记录化学发光值,实验结果如下:
表13实施例化合物对淋巴瘤细胞体外生长的抑制
表中对U937(组织细胞淋巴瘤细胞)、OCI-LY3(人弥漫大B细胞淋巴瘤细胞)、U2932(人弥漫大B细胞淋巴瘤细胞)、HT细胞(人混合淋巴瘤细胞)进行了细胞增殖实验分析。表格中显示本发明化合物对淋巴瘤细胞增殖具有抑制作用。其中,实施例2A对U937抑制活性最好,实施例13A对OCI-LY3细胞株和HT细胞的增殖抑制效果最好。表中“——”代表没有测数据。
实验27.PD小鼠模型
实验与分析方法:本实验使用C57BL/6品系的小鼠,分为5组,包括正常对照组(不注射MPTP),模型组(使用MPTP进行造模),以及3个小分子的治疗组(实施例13B,实施例25A,实施例25B)。造模的同时开始给药。造模持续5天,30mpk腹腔注射,给药持续15天,80mpk持续15天。终点时,心脏灌流后取脑置于PFA中浸泡固定,待固定完全后转移至蔗糖脱水,脱水完全后OCT包埋、切片,用于TH染色。检测指标为黑质TH阳性细胞数。阳性细胞越多,说明小分子对MPTP损伤的保护作用越好。
结果:每组12只小鼠,统计结果如图50所示,T检验,**:p<0.01,#:p<0.05。三种小分子与模型组相比,只有实施例13B组具有统计学差异,说明实施例13B具有很好的保护黑质神经元的效果。
实验28.新冠假病毒侵染细胞模型
实验与分析方法:首先使用HEK293T细胞培养只具有侵染能力而不具有复制能力的新冠假病毒。收集上清中的假病毒之后,使用表面具有ACE2受体的正常的HEK293T细胞作为侵染对象,进行假病毒侵染。被病毒侵染之后,HEK293T细胞中的线粒体会发生碎片化,可以由线粒体荧光染色(mitotracker red)来监测,说明细胞被假病毒所破坏,是为模型组。而在实验组,使用300nM的实施例13B提前与细胞孵育1小时再进行假病毒侵染,以检验小分子实施例13B对病毒侵染的保护作用。
结果:结果如图51显示,与正常对照组(不进行假病毒侵染)相比,模型组细胞中的线粒体碎片化明显,而实验组细胞中的线粒体则没有发生碎片化,说明小分子实施例13B对新冠假病毒的侵染具有很好的保护作用。
实验29.大鼠TBI急性颅脑创伤模型
实验与分析方法:本试验采用雄性SD大鼠,建立创伤性脑损伤(TBI)大鼠模型,随机分为:溶剂对照组(8只)、受试物实施例13B(80mpk,10只)、受试物实施例26(80mpk,9只)。TBI术后即刻开始给药,一天一次灌胃给药,连续7天。试验终点(第7天)采集脑组织检测海马区及损伤邻区细胞凋亡百分比。
结果:如图52所示流式结果显示,受试物实施例13B(80mpk)、受试物实施例26(80mpk)组大鼠的脑部早期凋亡率和总凋亡率均显著低于溶剂对照组(P<0.05)。在该试验体系下,受试物实施例13B、实施例26多次静脉给药可以明显改善急性颅脑创伤TBI大鼠神经功能损伤以及脑部细胞的凋亡,促进运动能力和神经功能的恢复。
实验30.对胰腺癌细胞的体外杀伤效果
实验与分析方法:我们使用Mia paca-2细胞作为研究对象。该细胞系为胰腺癌细胞系,被广泛地用于药敏实验。我们对11种小分子进行了药敏实验,最高浓度为10uM,3倍稀释,得到7个点的浓度梯度。使用celltiter glo进行细胞活性检测,并使用Graphpad prism进行剂量反应曲线的绘制。得出的药敏曲线如下图所示。
结果:药敏曲线如图53所示,IC50值越低则说明对该小分子越敏感。可以看到,Mia paca-2细胞对实施例26A的敏感度最高。
实验31.对DB动物过量排尿的抑制
实验与分析方法:瘦素受体基因(Obese Gene Receptor,OB-R)又称为糖尿病基因(Diabetes Gene,db),瘦素受体(leptin receptor,Lepr)与肥胖、高血压、糖尿病、脂质代谢紊乱等之间有密切关系。该疾病病程受基因背景影响很大。外用胰岛素无法控制血糖和肝脏葡萄糖异生水平的升高。利用基因编辑技术和胚胎注射技术构建Lepr基因突变小鼠。通过对该品系纯合子的血糖监测发现,其血糖水平与野生对照相比呈显著性差异,可用于II型糖尿病研究。该品系的小鼠尿量巨大,是正常小鼠的上百倍,因而可以摸拟尿频的病人。实验分为三个组,模型组(不给药,高尿量的发病鼠),对照组(不给药,不发病的正常鼠),以及实验组(给药实施例1A,80mpk,2天一次灌胃),对照组2只,另外两组3只。使用的小分子为实施例1A。我们从第8周开始,持续药物处理5周后,观察单笼饲养的小鼠在笼内的尿量情况。笼内尿湿面积越大,说明该笼小鼠的尿量越大。
结果:如图54的笼盒照片所示,三笼模型组小鼠笼内的尿湿面积最大,对照组小鼠笼内的尿湿面积几乎不可见,而给药组小鼠的笼内尿湿面积相比于模型组小鼠有明显的减少。由此说明,实施例1A有抑制尿频的功能。
实验32.对高脂饲喂的APOE突变小鼠的心血管炎症缓解作用。
实验与分析方法:购买6周龄雄性APOE-/-突变B6J小鼠和WT的B6J小鼠后先在饲养房间内适应2周。当小鼠8周龄时除了Vehicle组和WT组之外,其余组开始给高脂饲料(Research Diets,D12492)。当小鼠9周龄时,Control组给10%环糊精溶液,其余给药组开始分组经口灌胃不同的小分子化合物(实施例13B,实施例33,实施例36和实施例37),灌胃频率每2天一次。当高脂饲喂满16周后,处死APOE小鼠并解剖取材主动脉弓。将100mg/只固定质量的主动脉血管上皮组织进行MSD多因子检测,以确定各类炎症因子的表达量,包括IL10,IL-1β,和KC/GRO。
结果:如图55a至55c所示,三类炎症因子在小分子干预的作用下都有下降的趋势,而实施例13B的下降最为明显,说明所示小分子有抗心血管炎症的效果。
实验33.实施例13B对阈下催眠剂量戊巴比妥钠影响小鼠睡眠的药效实验
实验与分析方法:通过检测实施例13B单次经口灌胃给药,对阈下催眠剂量戊巴比妥钠诱导的ICR小鼠的入睡时间(睡眠潜伏期)及睡眠持续时间的影响,研究受试物是否具有催眠作用,并与阳性对照药地西泮进行药效学比较。小鼠适应性饲养后,根据体重挑选50只合格动物入组实验,采用Excel完全随机分组法分成5组:空白对照组、阳性对照组(地西泮)、受试物高、中、低剂量组,每组10只动物。各组动物按应给予药液及溶媒,给药1h后,各组动物腹腔注射戊巴比妥钠最大阈下剂量(首先进行预实验以确定戊巴比妥钠的最大阈下催眠剂量),记录每只动物的入睡时间(入睡潜伏期)、30min内入睡动物只数(翻正反射消失达1min以上者)、及每只鼠的睡眠持续时间。
结果:如图56a和56b所示,1mg/kg地西泮可明显缩短小鼠的睡眠潜伏期;与空白对照组相比,图示小分子均可缩短小鼠的睡眠潜伏期,且有一定量效关系,但并无统计学的显著性差异;此外,图示小分子缩短小鼠睡眠潜伏期的作用明显弱于1mg/kg地西泮。与空白对照组相比,阳性药组给予1mg/kg地西泮,明显延长小鼠的睡眠;与空白对照组相比,图示小分子可延长小鼠睡眠时间,但无统计学的显著性差异;此外,图示小分子延长小鼠睡眠时间的作用明显弱于1mg/kg地西泮。
在此说明书中,本发明已参照其特定的实施例作了描述。但是,很显然仍可以做出各种修改和变换而不背离本发明的精神和范围。因此,说明书应被认为是说明性的而非限制性的。

Claims (15)

  1. 一种基于菝契皂苷元结构的衍生物在制备治疗线粒体功能异常引起的相关联疾病的药物中的用途,其特征在于,所述的衍生物的结构式如通式(I)所示,
    所述的通式(I)所示的衍生物由以下片段A和片段B连接而成,
    其中,Z为NR1R2;R1和R2各自独立地为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、氨基、硝基、氰基、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基、苯基、苄基、C3-C14杂环基、C3-C14杂芳基,杂原子选自N、O、S的一个或多个;或者R1和R2一起形成三至八元环,所述的三至八元环具有一个或多个选自C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3-C14杂芳基、卤素、羟基、氨基、烷氧基、-CF3、-SF5或杂原子为硫、氧、NH或NRa的三至八元杂环的取代基,杂原子选自N、O、S的一个或多个;
    X为C(O)或S(O)2
    Y为C(Rd)(Re)、C(O)或S(O)2,Rd、Re独立地为氢或具有至少一个取代基的C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3-C14杂芳基,其中的取代基选自卤素、羟基、氨基、硝基、氰基、醛基、羧基、烷氧基、-CF3或-SF5,杂原子选自N、O、S的一个或多个,或者,Rd和Re一起形成三至八元环,所述的三至八元环具有一个或多个选自C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3-C14杂芳基、卤素、羟基、氨基、烷氧基、-CF3、-SF5或杂原子为硫、氧、NH或NRa的三至八元杂环的取代基,杂原子选自N、O、S的一个或多个;
    X2为O、S或NH;
    Ra独立地为氢或具有至少一个取代基的C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3-C14杂芳基,其中的取代基选自卤素、羟基、氨基、硝基、氰基、烷氧基、-CF3或-SF5,杂原子选自N、O、S的一个或多个;
    n为0至10的整数且n不为0,m为1,或者,n为0,m为1,或者,n为0至10的整数且n不为0,m为0;
    R3、R4a、R4b、R5a、R5b各自独立地为氢或选自卤素、取代的烷基、羟基、氨基;
    表示单键或者双键;
    各“*”独立地表示消旋、S或R构型。
  2. 根据权利要求1所述的用途,其特征在于,所述的衍生物的结构式如通式(II)所示,
  3. 根据权利要求1所述的用途,其特征在于,所述的衍生物的结构式如通式III所示,
  4. 根据权利要求1所述的用途,其特征在于,所述的衍生物的结构式如通式IV所示,
  5. 根据权利要求1所述的用途,其特征在于,所述的衍生物的结构式如通式V所示,
    其中,R6为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基、苯基、苄基,吡啶基,-CO烷基,-CO芳香基、-SO2烷基、-SO2芳香基、-CO2烷基、C2-C4(CO)链烯基、-CO2芳香基、-SO3H;
    L为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基;
    n为0至10的整数;
    n2为0,1,2,或3;
    m,m’独立地为1,2,3,或4;
    W1为C或NH;
    V1为C或NH;
    M为C、S、O或NH。
  6. 根据权利要求1所述的用途,其特征在于,所述的衍生物的结构式如通式VI所示,
    Y1为C(Rd)(Re)、C(O)或S(O)2,Rd、Re独立地为氢或具有至少一个取代基的C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3-C14杂芳基,其中的取代基选自卤素、羟基、氨基、硝基、氰基、醛基、羧基、烷氧基、-CF3或-SF5、杂原子选自N、O、S的一个或多个,或者,Rd和Re一起形成三至八元环,所述的三至八元环具有一个或多个选自C1-C10烷基、C3-C10环烷基、C6-C20芳基、或C3-C14杂芳基、卤素、羟基、氨基、烷氧基、-CF3、-SF5或杂原子为硫、氧、NH或NRa的三至八元杂环的取代基、杂原子选自N、O、S的一个或多个;
    L为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基;
    n为0至10的整数;
    n2为0,1,2,或3;
    n3为1至10的整数,
    m为0至10的整数。
  7. 根据权利要求1所述的用途,其特征在于,所述的衍生物的结构式如通式VII所示
    其中,R6、R7独立地为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基、苯基、苄基、吡啶基、-CO烷基、-CO芳香基、-SO2烷基、-SO2芳香基、-CO2烷基、C2-C4(CO)链烯基、-CO2芳香基、-SO3H;
    L为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、-NH2、硝基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基;
    W2为C或NH;
    V2为C、O、S或NH;
    n为0至10的整数;
    n1为1到10的整数;
    n2为0,1,2,或3。
  8. 根据权利要求1所述的用途,其特征在于,所述的衍生物的结构式如通式VIII所示
    Z1为氢、取代或未取代的C1-C10烷基,C1-C10烷基的取代基选自卤素、羟基、-NH2、硝 基、-CN、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C3-C6环烷基、C2-C4链烯基、C2-C4炔基、苯基、苄基、吡啶基、-CO烷基、-CO芳香基、-SO2烷基、-SO2芳香基、-CO2烷基、C2-C4(CO)链烯基、-CO2芳香基,-SO3H;
    W3为C、O、S或NH;
    n为0到10的整数;
    n4,n5,n6,n7独立地为1至4的整数。
  9. 根据权利要求1所述的用途,其特征在于,所述的衍生物的结构式中片段B为如下结构:
  10. 根据权利要求1所述的用途,其特征在于,所述的衍生物为以下化合物、以下化合物的非对映异构体混合物或以下化合物的对映异构体中的一种,



































  11. 根据权利要求1所述的用途,其特征在于,所述的化合物中的一个或多个氢原子为氘原子。
  12. 一种药物组合物在制备治疗线粒体功能异常引起的相关联疾病的药物中的用途,其特征在于,所述的药物组合物包括第一成分和药学上可接受的载体,或者第一成分、第二成分和药学上可接受的载体的组合,所述的第一成分为根据权利要求1至11任一项所述的用途中化合物、其药学上可接受的盐、立体异构体、互变异构体;
    所述的第二成分为附加治疗剂,所述的附加治疗剂包括抗抑郁药、抗狂躁药、帕金森病治疗药、阿尔兹默病治疗药或它们的组合。
  13. 根据权利要求12所述的用途,其特征在于,所述的药学上可接受的盐选自盐酸 盐、氢溴酸盐、硫酸盐、磷酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、1-萘磺酸盐、2-萘磺酸盐、乙酸盐、三氟乙酸盐、苹果酸盐、酒石酸盐、柠檬酸盐、乳酸盐、草酸盐、琥珀酸盐、富马酸盐、马来酸盐、苯甲酸盐、水杨酸盐、苯乙酸盐或扁桃酸盐的一种或多种;
    所述的附加治疗剂为吗氯贝胺、托洛沙酮、氟西汀、帕罗西汀、西酞普兰、舍曲林、文拉法辛、曲米帕明、曲唑酮、丙咪嗪、地昔帕明、氯米帕明、阿米替林、去甲替林、多塞平、马普替林、洛沙平、阿莫沙平、米氮平、丁螺环酮、氯美扎酮、坦度螺酮、碳酸锂、他克林、石杉碱甲、加兰他敏、多奈哌齐、力帆斯的明、美金刚、普拉克索、他利克索、罗匹罗尼,或它们的组合。
  14. 根据权利要求1至13中任一项所述的用途,其特征在于,所述的线粒体异常引起的相关联疾病,包括代谢类疾病、肿瘤、炎症、中枢神经系统性疾病。
  15. 根据权利要求14所述的用途,其特征在于,
    所述的代谢类疾病包括:高血糖、高血脂、高胆固醇、高低密度脂蛋白、低高密度脂蛋白,血管生成性病症、非酒精性脂肪肝性肝、脑血管意外、心肌梗死、动脉粥样硬化、冠心病、抗衰老、尿急尿频、I型糖尿病、慢性阻塞性肺病;
    所述的肿瘤包括:前列腺增生、韦格纳肉芽肿、肺结节病、白血病、淋巴瘤、胰腺癌、神经肿瘤;
    所述的炎症包括:外周神经炎、化疗诱导的外周神经炎、自体免疫疾病、与器官移植相关联的病状、流感病毒、冠状病毒的预防和感染治疗及其后遗症消除、急性呼吸窘迫综合征、炎性肠病、克罗恩氏病、溃疡性结肠炎、银屑病、视网膜脱离、色素性视网膜炎、黄斑变性、胰腺炎、特应性皮炎、类风湿性关节炎、脊椎关节炎、痛风、系统性红斑狼疮、干燥综合症、全省性硬皮病、抗磷脂综合征、血管炎、骨关节炎、自身免疫性肝炎、自身免疫性肝胆疾病、原发性硬发性胆管炎、肾炎、乳糜泻、自身免疫ITP、移植排斥、实体器官的缺血再灌注损伤、败血症、牙周炎、全身性炎症反应综合症、心肌炎、变应性疾病、哮喘、白细胞介素-I转化酶相关的发热综合征、白塞氏病;
    所述的中枢神经系统性疾病包括:皮克病、脊髓损伤修复、抑郁症、焦虑症、帕金森病、阿尔兹海默病、睡眠障碍、缺血性脑卒中、出血性脑卒中、肌肉萎缩性侧索硬化症、外伤性脑损伤、脑萎缩、亨廷顿病、精神分裂症、躁狂症、毒瘾戒断、多发性硬化症、改善睡眠、肌无力。
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