WO2022222686A1 - 木脂素衍生物、其制备方法及用途 - Google Patents

木脂素衍生物、其制备方法及用途 Download PDF

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WO2022222686A1
WO2022222686A1 PCT/CN2022/083003 CN2022083003W WO2022222686A1 WO 2022222686 A1 WO2022222686 A1 WO 2022222686A1 CN 2022083003 W CN2022083003 W CN 2022083003W WO 2022222686 A1 WO2022222686 A1 WO 2022222686A1
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alkyl
compound
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halogen
hydrogen
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沈建华
朱志远
李晓梅
王凯
刘佳
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中国科学院上海药物研究所
苏州青雅启瑞生物科技有限公司
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Priority to CN202280021312.3A priority Critical patent/CN117440947A/zh
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Definitions

  • the present invention relates to the field of medicinal chemistry, in particular, the present invention relates to lignan derivatives and their use as mitochondrial respiratory chain complex I inhibitors.
  • Mitochondrial respiratory chain complex I (NADH: ubiquinone oxidoreductase) is located in the inner membrane of mitochondria and is one of the most important protein complexes in the oxidative phosphorylation (OXPHOS) respiratory chain, which can transfer electrons from nicotinamide glands Purine dinucleotide (Nicotinamide Adenine Dinucleotide, NADH) is delivered to coenzyme Q (CoQ), coupled with four protons pumped from the mitochondrial matrix to the intermembrane space, forming a transmembrane proton gradient, driving adenine nucleoside triphosphate (ATP) )Synthesis. The protons pumped by complex I make up about 40% of the entire respiratory chain. Electron transfer through complex I and coupled proton pumping are the major pathways of energy production in normal cells.
  • tumor cells Unlike normal cells, tumor cells typically rely more on the glycolytic pathway for energy supply than the OXPHOS pathway for mitochondrial respiration.
  • OXPHOS pathway for mitochondrial respiration Unlike normal cells, tumor cells typically rely more on the glycolytic pathway for energy supply than the OXPHOS pathway for mitochondrial respiration.
  • research and development in recent years have found that in the process of tumorigenesis and the transformation of tumor cells, the way of energy metabolism is constantly changing, and some tumors have switched their energy supply mode to OXPHOS due to mutations in glycolysis-related genes. Changes in tumor cell energy metabolism provide new ideas for tumor therapy, and inhibiting the OXPHOS process in such tumors has become a unique therapeutic approach.
  • researchers at the University of Texas MD Anderson Cancer Center have developed a series of high-efficiency inhibitors of mitochondrial respiratory chain complex I, which can effectively inhibit the growth of acute leukemia cell lines in in vitro models.
  • IACS has good pharmacokinetic properties and oral bioavailability.
  • -10759 is currently in Phase I clinical trials in acute myeloid leukemia and solid tumors and lymphomas (Nature Medicine, 2018, 24, 1036-1046).
  • new research shows that the therapeutic resistance of tumor cells relying on the glycolytic metabolic pathway after some "targeted therapies" can be achieved through metabolic reorganization to OXPHOS-dependent shift, and OXPHOS inhibitors can effectively target Towards the development of metabolic recombination drug-resistant tumor cells.
  • the mitochondrial respiratory chain complex I inhibitor metformin effectively kills 5-fluorouracil-resistant colon cancer cells. (Oncotarget. 2015;6(39):41706-21.).
  • mitochondrial respiratory chain complex I inhibitors In addition to inhibiting the energy supply of the OXPHOS metabolic pathway, mitochondrial respiratory chain complex I inhibitors also exert antitumor effects through broader mechanisms, such as increased accumulation of intracellular reactive oxygen species (ROS) leading to cytotoxicity, inhibition of anaerobic inducible factor 1 Type alpha (HIF-1 ⁇ ) signaling, interference with mitochondrial membrane potential, activation of AMP-activated protein kinase (AMPK), etc. It can be seen that mitochondrial respiratory chain complex I inhibitors may also be used as broad-spectrum anti-tumor drugs or in combination with other mechanisms of anti-tumor drugs.
  • ROS reactive oxygen species
  • HIF-1 ⁇ anaerobic inducible factor 1 Type alpha
  • AMPK AMP-activated protein kinase
  • Senescent cells are a type of cells that permanently arrest the cell cycle but maintain high metabolic activity and continuously release the senescence-associated secretory phenotype (SASP). Senescent cells accumulate passively in organs during aging, leading to tissue dysfunction.
  • SASP senescence-associated secretory phenotype
  • the primary senescent cells generated by the "wear and tear" process of normal tissues will induce the senescence of adjacent cells, so that more senescent cells will be generated in the lesion site, and the cells with proliferative ability in the tissue will be lost, thereby leading to the occurrence of disease.
  • pancreatic beta cells can lead to diabetes
  • lung epithelial cells can lead to pulmonary fibrosis.
  • the inflammation caused by SASP secreted by senescent cells can also cause diseases, such as atherosclerosis, myocardial infarction, cardiomyocyte hypertrophy and other cardiovascular diseases and chronic obstructive pulmonary disease and other pulmonary diseases.
  • SASP-mediated extracellular matrix remodeling can also promote the process of tissue fibrosis, such as pulmonary fibrosis, liver fibrosis and kidney fibrosis.
  • Senescent cells are prevalent in many types of liver diseases, including viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease, liver fibrosis, liver cirrhosis, and hepatocellular carcinoma. Senescent hepatocytes, astrocytes and immune cells can promote liver disease progression through SASP, metabolic function, immune response and other pathways.
  • senescent cells also exist in other digestive organs such as the esophagus, stomach, bile duct, pancreas, and intestine, and play an important role in the occurrence and development of cholangitis, inflammatory bowel disease, and digestive tract tumors.
  • Aging is a major risk factor for neurodegenerative diseases, and the formation of various neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease, is related to cellular aging.
  • Senescent cells contribute to the formation and progression of neurodegenerative diseases by causing neuroinflammation, increasing oxidative stress, disrupting neuronal function, and hindering regeneration of the nervous system.
  • Senescent cell scavengers are a class of compounds that selectively remove senescent cells, prolong healthy lifespan, and enable individuals to reduce chronic disease distress in the aging process, including reducing tumorigenesis, prolonging median survival, reducing atherosclerosis, Osteoarthritis, cataracts, macular degeneration, cardiac hypertrophy, chronic kidney disease, chronic liver disease, pulmonary fibrosis, neurodegenerative diseases, lipid metabolism disorders, and sarcopenia and other aging changes.
  • senescent cell scavengers have entered clinical research on the treatment of aging-related diseases, such as the combination of dasatinib and quercetin in the treatment of idiopathic pulmonary fibrosis and entered the phase I clinical study, and UBX0101 in the treatment of osteoarthritis entered the phase II clinical study (Ageing Research Reviews, 2020, 66:101251.). Senescent cells accumulate in the microvessels of the diseased retina, and local removal of senescent cells with drugs can effectively prevent diseased angiogenesis in the retina and promote the repair of normal blood vessels (Cell Metab, 2021, 33:818.). The senescent cell scavenger UBX1325 is currently in clinical trials for the treatment of diabetic retinopathy and age-related macular degeneration.
  • OXPHOS In addition to the enhancement of OXPHOS in some subsets of tumor cells, studies have shown that the OXPHOS process in senescent cells is also enhanced. To maintain a high-level secretory phenotype, senescent cells must make significant metabolic changes, such as increased oxygen consumption and energy production, more active lipid breakdown, and high levels of ROS. Compared with glycolysis, senescent cells rely more on mitochondrial OXPHOS to provide energy, and the enhancement of mitochondrial function is an important determinant of SASP secretion by senescent cells. Therefore, in theory, targeting complex I of the mitochondrial respiratory chain and blocking OXPHOS is a reasonable approach to eliminate senescent cells.
  • mitochondria-targeted tamoxifen can selectively clear senescent cells by inhibiting mitochondrial respiratory chain complex I and reducing mitochondrial membrane potential (Cell Death & Differentiation, 2018, 26(2), 276-290.). Therefore, the application of mitochondrial respiratory chain complex I inhibitors to prevent or treat cell aging-related diseases may be an effective method.
  • arctigenin is a moderately potent and selective inhibitor of complex I of the mitochondrial respiratory chain.
  • Arctium aglycone has pharmacological effects such as anti-inflammatory, anti-infection, anti-metabolic disorder, and anti-nerve damage, and has a good effect on the treatment of cancer, atherosclerosis, diabetes, Alzheimer's disease and other aging-related diseases.
  • it is easily metabolized after oral administration, has poor pharmacokinetic properties and low bioavailability, which limits its application as an oral drug (Acta Pharmacologica Sinica, 2018, 39, 787–801.).
  • the purpose of the present invention is to provide a lignan derivative obtained by structural modification of arctigenin, which can effectively inhibit mitochondrial respiratory chain complex I.
  • the first aspect of the present invention provides a compound shown in formula I, its enantiomer, diastereomer, racemic mixture, deuterated compound and pharmaceutically acceptable salt,
  • Rx is selected from hydrogen, hydroxyl, halogen
  • R 0 is selected from hydrogen, halogen, C 1 -C 6 alkyl, hydroxy-substituted C 1 -C 6 alkyl, C 1 -C 6 haloalkyl;
  • R 2 and R 3 are each independently C 1- C 6 alkyl, C 1- C 6 haloalkyl, or R 2 and R 3 are linked together and together with the linked C to form an unsubstituted or substituted 5-8 membered heteroalkyl Cyclic group, said substitution means being substituted by one or more substituents selected from the group consisting of halogen, C 1 -C 6 alkyl;
  • R 4 is hydrogen or C 1- C 6 alkyl
  • each R 5 is each independently hydrogen, C 1- C 6 alkyl, -SO 2 R 13 , -COR 13 ;
  • Each R 9 is independently hydrogen, C 1- C 6 alkyl, C 6-10 aryl, 5-10-membered heteroaryl, and the aryl and heteroaryl can be optionally selected from 1-4 Substituted from the group consisting of halogen, hydroxy, -N(R 12 ) 2 , nitro, cyano, C 1- C 6 alkyl, C 1- C 6 haloalkyl, C 1- C 6 alkoxy base, 4-8 membered heterocyclic group;
  • Each R 11 is independently selected from C 1- C 6 alkyl and C 6-10 aryl optionally substituted with 1-4 groups selected from the group consisting of halogen, C 1- 6 alkyl, C 1-6 alkoxy;
  • R 8 , R 10 , and R 12 are each independently selected from hydrogen, C 1- C 6 alkyl
  • R 13 is C 1- C 6 alkyl
  • Y - is a halide ion, formate ion, acetate ion, trifluoroacetate ion or hydroxide ion;
  • the proviso is that when R 0 , R 4 , R x are all hydrogen and Ry is hydroxy, R 1 is not C 1 -C 6 alkoxy.
  • the halogen includes four atoms of fluorine, chlorine, bromine and iodine, and the halogen ion is a monovalent negative ion of fluorine, chlorine, bromine or iodine.
  • each R 5 is independently hydrogen or C 1- C 4 alkyl.
  • each R 9 is independently hydrogen or C 1- C 4 alkyl.
  • R 10 is hydrogen or C 1- C 4 alkyl.
  • each R 11 is independently C 1- C 4 alkyl or phenyl.
  • each R 12 is independently hydrogen or C 1- C 4 alkyl.
  • Ry is hydroxyl or Each R8 is independently hydrogen or C1 - C4 alkyl.
  • Rx is hydrogen, fluorine, chlorine or bromine.
  • R 0 is hydrogen, fluorine, chlorine, bromine, C 1 -C 4 alkyl, hydroxy-substituted C 1 -C 4 alkyl, and C 1 -C 4 haloalkyl.
  • R 4 is hydrogen or C 1- C 4 alkyl.
  • R 2 and R 3 are each independently a C 1- C 4 alkyl group, or R 2 and R 3 are connected together and formed together with the connected C R 6 and R 7 are each independently hydrogen, C 1- C 4 alkyl or halogen.
  • the compound has the following formula (IIa):
  • each substituent is the same as before, when R 0 is hydrogen, R 1 is not C 1 -C 6 alkoxy.
  • the compound has the following formula (IIb):
  • each R 8 is H.
  • R 1 is C 1 -C 4 alkoxy or -OLG; wherein, L is C 1-4 alkylene; G is phenyl or C 1 -C 4 haloalkyl; Ry is hydroxyl or -OP(O)(OH) 2 ; R 0 is hydrogen or C 1 -C 6 alkyl; R 2 and R 3 are each independently C 1 -C 4 alkyl.
  • the compound is selected from:
  • a second aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound described in the first aspect, its enantiomers, diastereomers, racemic mixtures, deuterated compounds and pharmaceutically acceptable salts;
  • a pharmaceutically acceptable carrier is selected from:
  • the third aspect of the present invention provides the compound described in the first aspect, its enantiomers, diastereomers, racemic mixtures, deuterated compounds and pharmaceutically acceptable salts or the compounds described in the second aspect
  • Uses of the pharmaceutical composition for the preparation of inhibitors of mitochondrial respiratory chain complex I; for the preparation of drugs for preventing and/or treating diseases associated with elevated mitochondrial respiratory chain complex I activity or expression; / or drugs for the treatment of diseases associated with enhanced mitochondrial oxidative phosphorylation; for the preparation of drugs for the prevention and/or treatment of diseases associated with cellular senescence; for the preparation of senescent cell scavengers; or for the preparation of prophylaxis and/or treatment cancer drugs.
  • the disease associated with enhanced mitochondrial oxidative phosphorylation is a tumor that partially or completely relies on the oxidative phosphorylation metabolic pathway to supply energy.
  • the disease associated with enhanced mitochondrial oxidative phosphorylation is a disease associated with cellular senescence.
  • the tumor is selected from acute myeloid leukemia, glioma, lymphoma, pancreatic cancer, uterine cancer, breast cancer, non-small cell lung cancer, and hepatocellular carcinoma.
  • the tumor is acute myeloid leukemia or glioma.
  • the disease related to cell senescence is selected from: organ fibrosis disease, chronic lung disease, chronic kidney disease, chronic liver disease, osteoarthritis, neurodegenerative disease, inflammatory bowel disease, atherosclerosis , glaucoma, cataract, macular degeneration, diabetes, diabetic retinopathy, pigmentation, sarcopenia.
  • the disease related to cell senescence is an organ fibrosis disease selected from the group consisting of pulmonary fibrosis, liver fibrosis, and renal fibrosis.
  • the disease related to cellular senescence is idiopathic pulmonary fibrosis.
  • the disease related to cell senescence is liver fibrosis.
  • the disease related to cellular senescence is osteoarthritis.
  • the cell aging-related disease is a neurodegenerative disease selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis.
  • the disease related to cellular senescence is Alzheimer's disease.
  • the disease related to cell senescence is inflammatory bowel disease, selected from ulcerative colitis and Crohn's disease.
  • the present invention also provides a preparation method of the compound represented by the general formula (I), the method is selected from the following route 1 or route 2, specifically, depending on the type of the five-membered lactone ring.
  • the protecting group Pg is a group that makes it possible to protect reactive functional groups, such as hydroxyl or amines, on the one hand during the synthesis and, on the other hand, to restore the reactive functional groups to their integrity at the end of the synthesis.
  • reactive functional groups such as hydroxyl or amines
  • Scheme 1 is applicable to R 0 is selected from hydrogen, halogen, C 1 -C 6 alkyl, hydroxy-substituted C 1 -C 6 alkyl, C 1 -C 6 haloalkyl; R 4 is selected from hydrogen; other substituent definitions Compounds as previously described.
  • Scheme 2 is applicable to compounds as R 0 is selected from hydrogen, R 4 is selected from C 1 -C 6 alkyl, and the other substituents are as defined above.
  • the second synthetic route starts from the intermediate compound 5.
  • the hydroxyl group of compound 5 is oxidized by an oxidant to obtain the corresponding aldehyde, and then the Grignard reagent reacts with the aldehyde group to free the hydroxyl group to obtain the corresponding compound 11.
  • Compound 11 undergoes a transesterification reaction under acid catalysis to the key intermediate butyrolactone compound 12.
  • a substituted benzyl compound is introduced at the ⁇ -position of the ester carbonyl group of compound 12 by hydrogen extraction with an organic strong base to obtain compound 13, and the phenolic hydroxyl group of compound 13 is deprotected to obtain a compound of general formula (III).
  • Figure 1 shows the reaction time of mice in the hot plate experiment.
  • Figure 2 shows pictures of mouse joint pathological morphology and OA score.
  • Fig. 3 shows the p16 positive rate of articular cartilage immunohistochemistry.
  • Figure 4 shows the changes in body weight after administration of IPF mice.
  • Figure 5 shows the changes in survival rate of mice after administration.
  • Figure 6 shows Masson staining and pathological scoring of mouse lung tissue sections.
  • Figure 7 shows the level of p16 gene, a marker of aging in mouse lung tissue.
  • Figure 8 shows IL-6 gene levels in mouse lung tissue SASP.
  • Figure 9 shows the effect of compound A4-1a on the latency of transgenic AD mice in the water maze test.
  • Figure 10 shows the effect of compound A4-1a on the number of crossed platforms in transgenic AD mice.
  • Figure 11 shows the effect of compound A5 on the content of hydroxyproline in the liver tissue of mice with carbon tetrachloride-induced liver fibrosis.
  • Figure 12 shows the effect of compound A5 on the positive rate of Masson staining in liver of mice with hepatic fibrosis.
  • Figure 13 shows the effect of compound A5 on the disease activity index of oxazolone-induced enteritis mice.
  • Figure 14 shows the effect of Compound A5 on colon pathology in mice with oxazolone-induced enteritis.
  • Figure 15 shows the drug concentration/time curve in plasma, liver and colon tissue of Compound A4-1a and Arctigenin after oral administration of 30 mg/kg to mice.
  • Figure 16 shows the drug concentration/time curve of prodrug A5 (A) and prototype A4-1a (B) in plasma and tissue after compound A5 was orally administered to mice at 30 mg/kg.
  • C1-C6 means having 1, 2, 3, 4, 5 or 6 carbon atoms
  • C1-C8 means having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, and so on.
  • 3-8 membered means having 3, 4, 5, 6, 7 or 8 ring atoms
  • 5-7 membered means having 5, 6 or 7 ring atoms, and so on.
  • aromatic ring and “aryl group” are defined as monocyclic and bicyclic ring systems consisting of a specific number of carbon atoms and complying with Hückel's rule, including but not limited to benzene ring and naphthalene ring.
  • heteroaryl group are defined as having a specific number of ring atoms and containing 1, 2, 3 or 4 heteroatoms (selected from N, O, S), and complying with Hückel Regular monocyclic and bicyclic ring systems, including but not limited to pyridine, pyrrole, imidazole.
  • aryloxy is defined as a group formed by connecting an oxygen to an aromatic group, including but not limited to Ph-O-.
  • heterocyclic group is defined as a saturated or unsaturated non-aromatic cyclic group consisting of a specific number of carbon atoms, and containing 1, 2, 3 or 4 heteroatoms (selected from N, O, S), including but not limited to morpholine, piperazine.
  • alkyl group and the similar “alkoxy group” are branched and straight chain hydrocarbon groups and hydrocarbon oxygen groups with a specific number of carbon atoms, and representative examples include but are not limited to methyl groups , methoxy, ethyl, ethoxy, n-propyl, n-propoxy, isopropyl, isopropoxy.
  • haloalkyl group refers to a group formed by partially or completely substituting a hydrogen atom in an “alkyl group” having a specific number of carbon atoms by a “halogen atom”.
  • the "halogen atom” includes fluorine, chlorine, bromine and iodine.
  • the substitution is monosubstitution or polysubstitution
  • the polysubstitution is disubstitution, trisubstitution, tetrasubstitution, or pentasubstitution.
  • the disubstituted refers to having two substituents, and so on.
  • the pharmaceutically acceptable salts of the present invention may be salts of anions with positively charged groups on the compounds of formula I.
  • Suitable anions are chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, tosylate, tartrate, fumarate acid, glutamate, glucuronate, lactate, glutamate or maleate.
  • salts can be formed from cations with negatively charged groups on compounds of formula I. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium, such as tetramethylammonium.
  • “pharmaceutically acceptable salt” refers to the salts formed by the compound of formula I with an acid selected from the group consisting of hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, Nitric acid, methanesulfonic acid, sulfamic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid , Glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic
  • the compound of general formula I of the present invention or its pharmaceutically acceptable salt is obtained by distillation, crystallization or recrystallization from water or organic solvent, and the compound may contain the solvent molecules used. In addition, different crystallization conditions may result in different crystalline forms of the compounds. Therefore, compounds of general formula I or pharmaceutically acceptable salts thereof containing various stoichiometric amounts of crystallization solvents and all crystalline forms are within the scope of the present invention.
  • the compound represented by the general formula (I) is usually in the form of a pharmaceutical composition when it is used to prepare a medicine, and the pharmaceutical composition comprises the compound of the general formula (I) and one or more pharmaceutically acceptable excipients;
  • the pharmaceutically acceptable adjuvants are pharmaceutically acceptable carriers, excipients, sustained-release agents, odorants, flavoring agents, etc.; in the pharmaceutical composition, the compound represented by the general formula (I) is used as the active component, Its weight accounts for 0.1-99.9% of the total weight of the pharmaceutical composition, and the rest are pharmaceutically acceptable auxiliary materials; Suspensions, aerosols, dry powders, etc., and can be stored in suitable sterile implements and delivery devices.
  • the "effective therapeutic dose” means that, compared with the subject who did not receive the dose, the subject who received the dose was cured, improved, effectively prevented or the incidence of the disease or side effects was significantly reduced; in addition, It also includes an effective dose to enhance normal physiological function.
  • the compound represented by the general formula (I), its salt and its pharmaceutical composition can be used for humans and animals; the route of administration includes oral administration, inhalation, transdermal absorption, injection, etc.; in the present invention, the preferred administration The route is oral administration; when the compound represented by the general formula (I), its salt and its pharmaceutical composition are used in the preparation of medicines; the dosage and frequency of administration should be determined according to the doctor's advice.
  • the compound represented by the general formula (I), its salt and its pharmaceutical composition can be used in combination with other drugs when used for the prevention or treatment of cancer and aging-related diseases;
  • the drugs include but are not limited to anti-tumor drugs, treatment Tissue fibrosis drugs, drugs for the treatment of osteoarthritis.
  • the "acid”, “base”, “oxidizing agent” and “reducing agent” have the same meaning as those familiar to those skilled in the art, and those skilled in the art can use the specific reaction substrates and reaction conditions according to the difference Make appropriate changes and adjustments.
  • Bn benzyl
  • DCM dichloromethane
  • DIPEA N,N-diisopropylethylamine
  • DMAP 4-dimethylaminopyridine
  • DMF N,N-dimethylformamide
  • THF tetrahydrofuran
  • LDA lithium diisopropylamide
  • NaHMDS sodium bis(trimethylsilyl)amide.
  • (S)-4-benzyl-2-oxazolidinone (B1-2) (8.0 g, 45.0 mmol) in THF was added dropwise ( 100 mL), add anhydrous LiCl (2.10 g, 50.0 mmol) at one time, continue stirring for 1 h, then warm to room temperature, and continue stirring for 4 h.
  • the reaction solution was concentrated under reduced pressure to about 50 mL, diluted with 100 mL of ethyl acetate, followed by 10% Wash with sodium bicarbonate, 5% potassium hydrogen sulfate, saturated sodium chloride solution, and dry with anhydrous sodium sulfate.
  • step B2-1 The product of the previous step B2-1 (15.8 g, 43 mmol) was dissolved in 500 mL of anhydrous THF, and NaHMDS (32 mL, 64 mmol, 2 mol/L in THF) was added dropwise at -78 ° C. Stirring for 1 hour, tert-butyl bromoacetate ( 12.6mL, 85mmol), continue to stir at this temperature for 5h.
  • step B5-3b The product of the previous step B5-3b (1.04g, 4mmol) was dissolved in 20mL of anhydrous THF, tetrahydroaluminum lithium solution (2.5M/L in THF) (2.5ml, 10mmol) was added dropwise under an ice bath, and the ice bath was stirred for 30 After minutes, the temperature was slowly raised to room temperature for 2 hours.
  • tetrahydroaluminum lithium solution 2.5M/L in THF
  • Mitochondria were extracted from mouse heart tissue.
  • Heart tissue was added with 200-250mL of solution A (0.22M mannitol (mannitol), 0.07M sucrose (sucrose), 0.02M HEPES, 2mM Tris-HCl, 1mM EDTA, pH 7.2, 0.4%BSA), the tissue was minced, and then Wash 3 more times with solution A to remove blood and connective tissue. Using a glass homogenizer, homogenize the tissue about 30 times. Suspend at 3000g for 1.5 minutes, take the supernatant for use, add protease inhibitor to precipitate solution A, resuspend, re-centrifuge, collect the supernatant, and mix the two supernatants for the next step.
  • solution A 0.22M mannitol (mannitol), 0.07M sucrose (sucrose), 0.02M HEPES, 2mM Tris-HCl, 1mM EDTA, pH 7.2, 0.4%BSA
  • NADH activity assay of Complex I 2.5mL reaction solution was prepared: 50mM HEPES, 5mM MgCl 2 pH 7.5 was added with 30 ⁇ g mitochondrial protein in the above process. Then add the compound to be detected and equilibrate for 5 minutes, then dispense into each well, and finally add NADH (50 ⁇ M) rapidly, read once every 30 sec at 340 nM, and carry out a total of 15 minutes of reaction. Calculation: Calculate the NADH reaction rate in different compounds, then calculate the inhibition rate (compound reaction rate/control reaction rate*100), and finally calculate the IC 50 value of the compound.
  • the compound of the present invention has a complex I inhibitory activity at a micromolar to nanomolar level, and can be used to prepare a drug for the treatment of diseases caused by the abnormal function of the complex I of the mitochondrial respiratory chain. .
  • OCI-AML3 cells are suspension cells, cultured in RPIM1640+10%FBS+1%PS medium at 37°C, 5%CO 2 .
  • OCI-AML3 was seeded in a 96-well plate at 20,000 cells/well, and different concentrations of compounds were added at the same time (40 ⁇ M was the highest concentration, diluted by 1/4, a total of 7 concentrations), and after 3 days of co-incubation, 10 ⁇ l was added to each well. After incubation for 2 hours, the OD450 was detected by a microplate reader, and then the effect of the compound on the cell viability was calculated, and the IC50 value was calculated. 2.2 Evaluation results
  • the compound of the present invention has a good effect of inhibiting the activity of OCI-AML3 cells, and can be used to prepare a drug for the treatment of acute myeloid leukemia.
  • NB-1 cells are suspension cells, cultured in RPIM1640+10%FBS+1%PS medium at 37°C, 5%CO 2 .
  • NB-1 was seeded in a 96-well plate at 20,000 cells/well, and different concentrations of compounds were added at the same time (40 ⁇ M was the highest concentration, diluted by 1/4, a total of 7 concentrations), and after 3 days of co-incubation, 10 ⁇ L was added to each well. After incubation for 2 hours, the OD450 was detected by a microplate reader, and then the effect of the compound on the cell viability was calculated, and the IC50 value was calculated.
  • Table 8 represents the inhibitory activity of compounds on NB-1 cells
  • the compound of the present invention has a good effect of inhibiting the activity of NB-1 cells, and can be used to prepare a drug for the treatment of glioma.
  • WI-38 cells were cultured in DMEM (high glucose, 4.5 g/L) medium (added with 10% FBS, 1% PS).
  • WI-38 cells were seeded in 96-well plates at 20,000 cells/well, and 200 nM doxorubicin was added to each well overnight to incubate for 3 days, and then replaced with normal culture.
  • WI-38 cells can be successfully induced to senesce by incubating with 200nM doxorubicin for 1 day, and then replaced with 200nM doxorubicin for 3 days; then different concentrations of compounds (40 ⁇ M is the highest concentration, diluted according to 1/4, a total of 7 cells) were added.
  • MOL-Y4 cells were cultured in MEM medium (added with 10% FBS, 1% PS).
  • MOL-Y4 cells were seeded in 96-well plates at 20,000 cells/well, and 100 nM doxorubicin was added to each well overnight to incubate for 3 days, and then replaced with normal culture.
  • the MOL-Y4 cells can be successfully induced to senesce by incubating with 100nM doxorubicin for 1 day, and then replaced with 100nM doxorubicin for 3 days; then different concentrations of compounds (40 ⁇ M is the highest concentration, diluted according to 1/4, a total of 7 cells) can be successfully induced.
  • BCL-2 inhibitor ABT263 is a positive control.
  • the compounds of the present invention can effectively inhibit the viability of senescent cells. Since senescent cells do not have the ability to replicate, the reduction in cell viability represents drug-induced apoptosis, that is, elimination.
  • the compound of the invention has a better effect on removing senescent cells than the existing senescent cell scavenger ABT263, and can be used for preparing medicines for treating senescence-related diseases.
  • Compound A4-1a (2 mM, 50 ⁇ L/mice) was administered to the administration group after recovery of the mice on the day after surgery, and a solvent (physiological saline) was administered to the other groups. It is then administered every other day for 8 weeks.
  • Fig. 1 shows the reaction time of mice in the hot plate test
  • Fig. 2 shows the pictures of joint pathological morphology and OA score of mice
  • Fig. 3 shows the p16 positive rate of articular cartilage immunohistochemistry.
  • the compound A4-1a of the present invention can significantly reduce the reaction time of the osteoarthritis mice in the hot plate test, indicating that it is helpful for the recovery of the exercise ability of the mice.
  • A4-1a can restore the smoothness and integrity of the damaged articular cartilage surface, reduce the joint histopathological score in mice, and reduce the expression of the aging marker p16 in the cartilage, indicating that compound A4-1a can effectively clear the damage induced senescent cells, and improve the pathological morphology of bone and joint.
  • mice Male C57 mice, 24-28g/mice, 70 rats.
  • Bleomycin-induced pulmonary fibrosis model and administration 65 C57 mice, bleomycin at 2U/kg, after anesthesia, administered by oropharyngeal inhalation for IPF modeling, before and after administration
  • the corresponding body weight of each mouse was measured, and the mice with obvious weight loss were selected as the standard for successful modeling, and 20 mice with successful modeling were selected and divided into 2 groups (respectively: solvent group - given solvent, A4-1a-30 group - oral administration of A4-1a 30mg/kg/day, A4-1a-10 group - oral administration of A4-1a 10mg/kg/day), and 5 C57 mice were selected in addition as a normal mouse control. Dosing was started on day 5 and administered for 14 days. The body weight and survival rate of the mice were recorded daily.
  • mice On the 15th day after administration, the mice were weighed after anesthesia, and the mice were sacrificed. The lung tissue of the mice was removed and weighed. The calculation method of the mouse lung index: weight of mouse lung tissue/weight of mouse *1000; The middle 1/3 of lung tissue was taken out and fixed in 4% paraformaldehyde for subsequent paraffin sectioning and Masson staining.
  • Masson staining of mouse lung tissue After paraffin-embedding mouse lung tissue according to conventional methods, the sections were subjected to Masson staining. The specific procedure is as follows: after section deparaffinization, hematoxylin staining for 2 minutes, distilled water for 2-3 seconds, differentiation solution for 2-3 seconds, distilled water for 2-3 seconds, blue-return solution for 2-3 seconds, distilled water for 2-4 minutes, and magenta staining 6 min, distilled water 2-3 sec, phosphomolybdic acid 2-3 sec, aniline blue 2 min, 1% acetic acid 2-3 sec, 100% ethanol 2 min, repeat 100% ethanol 2 min, xylene 2 min, repeat two Toluene for 2 minutes, and the slides were mounted with neutral gum.
  • Figure 4 shows the changes in body weight of IPF mice after administration
  • Figure 5 shows the changes in survival rate of mice after administration
  • Figure 6 shows Masson staining and pathological scores of mouse lung tissue sections
  • Figure 7 shows mouse lungs Tissue senescence marker p16 gene level
  • Figure 8 shows IL-6 gene level in mouse lung tissue SASP.
  • the compound A4-1a of the present invention can significantly slow down the weight loss of IPF mice, improve the survival rate of mice, reduce the degree of fibrosis in lung tissue sections (Masson staining score), and reduce the level of aging markers p16 and p16 in lung tissue.
  • the aging-related secretory phenotype of IL-6 indicates that the compound has a very significant effect on improving or treating the aging-related disease IPF. 7.
  • APP/PS1 transgenic Alzheimer's disease (AD) model mice are used. These transgenic mice highly express the chimeric murine/human Swedish mutant APP (Mo/HuAPP695swe) and the human-derived presenilin 1 protein (presenilin, PS1-dE9) with the deletion of the ninth exon. Rats showed significant A ⁇ deposition at 6 months and impaired spatial memory at 7 months. Therefore, the present invention adopts six-month-old APP/PS1 transgenic AD mice, and after three months of intragastric administration (A4-1a, 30 mg/kg/day), the Morris water maze test is used to detect the effect of A4-1a on transgenic mice. The memory impairment of mice, and the negative mice of this type of mice were used as the control group to evaluate the therapeutic effect of A4-1a on Alzheimer's disease.
  • AD Alzheimer's disease
  • APP/PS1 double transgenic AD model mice were bred.
  • the APP/PS1 gene sequence of the mice was identified by using tail clipping of the mice and then PCR.
  • Non-transgenic mice were used as negative control mice in the experiments. Mice were housed under standard conditions (12/12 hour light-dark cycle, adequate water and food, constant temperature of 22°C, 60% humidity).
  • AD model mice At the age of 6 months, 20 transgenic mice were randomly divided into 2 groups (transgenic solvent group, transgenic A4-1a-30 mg/kg/day dose group), 10 non-transgenic mice. Transgenic mice served as a negative control group. A4-1a was dissolved in 2% Tween 80 and administered by gavage for 90 days, and then the behavioral test (Morris water maze test) was started.
  • mice were trained three times a day for a period of 8 days. After training the mice to face the pool wall in the water for 60 seconds to find the platform position, the mice stayed on the platform for 15 seconds to help it memorize the platform position. During this period, the time it took for the mice to find the platform was recorded as the latency period.
  • the mice performed a platform-seeking experiment, the platform submerged in the water was removed, and then the mice were left in the pool for 90 seconds to find the platform, and the number of times the mice crossed the platform was recorded as their memory. The evaluation index, the more times, the better the memory. All animal experimental operations strictly abide by the "Administrative Regulations on Laboratory Animals".
  • transgenic mice T-V
  • NT-V non-transgenic mice
  • AD model AD model is correct.
  • Transgenic mice (T-A4-1a-30) administered orally with A4-1a at 30 mg/kg/day had significantly shorter latency than solvent-treated transgenic mice, and the number of crossing platforms was significantly higher than that of solvent-treated transgenic mice, indicating that A4 -1a reverses memory impairment in transgenic mice.
  • mice 400 uL of carbon tetrachloride (CCl 4 ) was added to 3.6 mL of olive oil, mixed well, and then intraperitoneally injected at 0.1 mL/20 g, twice a week for six weeks. After six weeks, 30 mice were divided into 3 groups, 10 mice in each group, and were given normal saline by gavage, A5 dose of 1 mg/kg and A5 dose of 3 mg/kg. In addition, 10 mice that were not induced were given physiological saline as a control group. Mice were sacrificed after continuous administration for 3 weeks, and the livers were weighed, fixed and embedded, and the remaining liver tissues were cryopreserved for detection of hydroxyproline content.
  • CCl 4 carbon tetrachloride
  • Masson staining of mouse liver tissue After paraffin-embedding mouse liver tissue according to conventional methods, the sections were subjected to Masson staining. The specific procedure is as follows: after section dewaxing, hematoxylin staining for 2 minutes, distilled water for 2-3 seconds, differentiation solution for 2-3 seconds, distilled water for 2-3 seconds, blue-return solution for 2-3 seconds, distilled water for 2-4 minutes, and magenta staining 6 min, distilled water 2-3 sec, phosphomolybdic acid 2-3 sec, aniline blue 2 min, 1% acetic acid 2-3 sec, 100% ethanol 2 min, repeat 100% ethanol 2 min, xylene 2 min, repeat two Toluene for 2 minutes, and the slides were mounted with neutral gum.
  • hydroxyproline in mouse liver tissue was detected by using a hydroxyproline detection kit (Nanjing Jiancheng Bioengineering Institute).
  • the brief introduction is as follows: Weigh 40 mg of liver tissue, add 0.5 mL of alkaline lysis solution, and hydrolyze 20 mg at 95°C. minutes, then add 5 ⁇ L of pH indicator, mix well, then add 0.5ml of pH-adjusting solution A, it turns red after mixing, and slowly add pH-adjusting solution B until the red disappears and turns yellow-green.
  • the liver of mice induced by CCl 4 and administered with solvent contained hydroxyproline (HYP) higher than that of normal control mice, which was statistically significant.
  • compound A5 administered by intragastric administration of 1 mg/kg/day and 3 mg/kg/day could significantly reduce the increase of hydroxyproline content induced by CCl 4 , with statistical significance.
  • Modeling and administration 3% oxazolone was dissolved in a solvent (acetone: olive oil, 1:4), after the mice were anesthetized, the hair on the shoulders of the mice was removed, and then 150uL was added to a mass fraction of 3 % Oxazolone. On the 8th day after treatment, 1% Oxazolone was dissolved in a solvent (ethanol: sterile water, 1:1), and mice were anesthetized, and 150 uL of 1% Oxazolone was administered rectally to make a model. The model mice were divided into two groups with 10 mice in each group.
  • mice One group was given compound A5 by gavage at 1 mg/kg/day, the other group was given normal saline, and the other group of 10 mice without modeling was used as the control group. From the day of modeling, the body weight of the mice was recorded every day, and the feces of the mice were collected on the third day after rectal administration to observe the softness of the feces and whether there was bleeding. Then the mice were sacrificed, and the distal 3 cm of the mouse colon was taken for fixation.
  • mice enteritis disease activity index score according to the degree of weight loss in mice (0: normal; 1: 1–5%; 2: 6–10%; 3: 11–18%; 4: >18%), the degree of fecal softness (0: normal; 2: soft; 4: diarrhea), the degree of fecal hemorrhage (0: normal; 2: occult blood; 4: bloody stool), and then the scores were added and divided by three.
  • Mouse colon inflammatory index calculation method According to the integrity of mouse colon mucosa (0: normal; 1: mucosal damage; 2: submucosal damage; 3: muscle damage), the degree of inflammatory cell infiltration (0: normal; 1: massive inflammation Cell mucosal infiltration; 2: inflammatory cell infiltration into the submucosa; 3: inflammatory cell infiltration into the muscularis), the degree of edema of the colon (0: normal; 1: mild edema; 2 moderate edema; 3 moderate edema) .
  • the disease activity index of Oxazolone-induced enteritis model mice was significantly higher than that of the normal group, and compound A5 (1 mg/kg/day) administered by gavage could significantly reduce the disease activity index of enteritis.
  • the colonic pathological score of Oxazolone-induced enteritis model mice was significantly higher than that of normal control mice, and intragastric administration of A5 (1 mg/kg/day) could significantly reduce the colonic pathological score.
  • Figure 15 shows the drug concentration/time curve in plasma, liver and colon tissue of Compound A4-1a and Arctigenin after oral administration of 30 mg/kg to mice.
  • BNPP sodium heparin and bis(4-nitrophenyl) phosphate
  • Figure 16 shows the drug concentration/time curve of prodrug A5 (A) and prototype A4-1a (B) in plasma and tissue after compound A5 was orally administered to mice at 30 mg/kg.
  • the phosphate prodrug A5 can efficiently metabolize the prototype compound A4-1a in vivo after oral administration, and has a good drug concentration in plasma and various tissues. Therefore, it is used to prevent or treat the diseases of the present invention. It also has potential application value in the drug.
  • the present invention is based on the structural modification of arctigenin (Arctigenin) to obtain the compound of general formula I and produces unexpected effects, either improving the activity of Complex I, or improving the activity of inhibiting tumor cells, or improving the activity of removing senescent cells, Or improve the oral pharmacokinetic properties, or show a very significant pharmacodynamics in animal models of disease, so in the prevention and / or treatment and mitochondrial respiratory chain complex I activity or expression increased, or mitochondrial oxidative phosphorylation It has good application potential in diseases related to potentiation.

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Abstract

本发明公开了一种木脂素衍生物、其制备方法及用途，所述木脂素衍生物结构如式I所示，式中，各取代基的定义如说明书和权利要求书中所述。本发明的木脂素衍生物，能够作为线粒体呼吸链复合物I抑制剂，抑制线粒体的氧化磷酸化作用及ATP的生成，用于预防和/或治疗与线粒体呼吸链复合物I活性或表达升高、或者线粒体氧化磷酸化作用增强相关的疾病。

Description

木脂素衍生物、其制备方法及用途 技术领域
本发明涉及药物化学领域，具体地，本发明涉及木脂素类衍生物及其作为线粒体呼吸链复合物I抑制剂的用途。
背景技术
线粒体呼吸链复合物I(NADH:泛醌氧化还原酶)位于线粒体的内膜，是氧化磷酸化(oxidative phosphorylation；OXPHOS)呼吸链中最重要的蛋白复合体之一，可以将电子从烟酰胺腺嘌呤二核苷酸(Nicotinamide Adenine Dinucleotide，NADH)传递至辅酶Q(CoQ)，同时偶联四个质子从线粒体基质泵出至膜间隙，形成跨膜质子梯度，驱动腺嘌呤核苷三磷酸(ATP)的合成。复合物I泵出的质子占整个呼吸链的40％左右。通过复合物I进行的电子传递及偶联的质子泵出是正常细胞中能量产生的主要途径。
与正常细胞不同，肿瘤细胞通常更依赖于糖酵解途径进行能量供应，而非线粒体呼吸OXPHOS途径。不过近年的研发发现，在肿瘤的发生过程和肿瘤细胞的转化过程中，能量代谢方式是在不断变化的，有部分肿瘤由于糖酵解相关基因的突变而将能量供应方式转向OXPHOS。肿瘤细胞能量代谢的改变为肿瘤的治疗提供了新思路，抑制这类肿瘤中的OXPHOS过程成为一条独特的治疗途径。美国德州大学安德森癌症中心研究人员开发了一系列高效线粒体呼吸链复合体I抑制剂，体外模型中能有效地抑制急性白血病细胞系的生长，其中药动学性质和口服生物利用度表现良好的IACS-10759目前正处于急性髓性白血病以及实体瘤和淋巴瘤的I期临床试验中(Nature Medicine,2018,24,1036-1046)。此外，新的研究表明，依靠糖酵解代谢途径的肿瘤细胞在一些“靶向疗法”后发生的治疗抗性可通过代谢重组至OXPHOS依赖性的转变来实现，而OXPHOS抑制剂能有效地靶向发生代谢性重组耐药肿瘤细胞。例如，线粒体呼吸链复合物I抑制剂二甲双胍能有效地杀死5-氟尿嘧啶耐药的结肠癌细胞。(Oncotarget.2015；6(39):41706-21.)。
除了抑制OXPHOS代谢途径的能量供应，线粒体呼吸链复合体I抑制剂还通过更广泛的机制发挥抗肿瘤作用，例如增加细胞内活性氧簇(ROS)的蓄积导致细胞毒性、抑制厌氧诱导因子1型alpha(HIF-1α)信号、干扰线粒体膜电位、激活AMP-活化的蛋白激酶(AMPK)等。可见，线粒体呼吸链复合体I抑制剂还可能用作广谱的抗肿瘤药物或者与其他机制的抗肿瘤药物联用。
另一方面，研究表明，许多与年龄有关的疾病都与细胞衰老有关，包括早衰综合征、心血管疾病、骨关节炎、癌症、神经退行性疾病、慢性肾病、慢性阻塞性肺病、特发性肺纤维化以及II型糖尿病、皮肤衰老、色斑沉积等病变。衰老细胞是一类细胞周期永久停滞、却保持着高代谢活性、并持续释放衰老相关分泌表型(SASP)的细胞。衰老细胞在衰老过程中被动地积聚在器官中，导致组织功能障碍。正常组织的“磨损”过程产生的初级衰老细胞会诱导临近细胞发生衰老，使更多衰老细胞在病灶部位产生，使组织中具有增殖能力的细胞缺失，从而导致疾病的发生。例如胰岛β细胞的缺失会导致糖尿病，肺上皮细胞的缺失会导致肺纤 维化。同时，衰老细胞分泌的SASP引起的炎症也会引起疾病，如动脉粥样硬化、心肌梗塞、心肌细胞肥大等心血管病变以及慢性阻塞性肺病等肺部病变。另外，SASP介导的细胞外基质重塑也可在组织纤维化进程中起促进作用，如肺纤维化、肝纤维化和肾纤维化等。衰老细胞普遍存在于众多类型的肝病组织中，包括病毒性肝炎、酒精性肝病、非酒精性脂肪肝、肝纤维化、肝硬化以及肝细胞癌。衰老的肝细胞、星形细胞和免疫细胞可通过SASP、代谢功能、免疫反应等途径促进肝病进展。同样，衰老细胞也存在于食道、胃、胆管、胰腺、肠道等其他消化器官中，在胆管炎、炎症性肠病、消化道肿瘤等病变发生发展过程中起到了重要作用。衰老是神经退行性疾病的主要风险因素，包括阿尔兹海默症、帕金森症在内的多种神经退行性疾病的形成与细胞衰老有关。衰老细胞通过引起神经炎症、增加氧化应力、破化神经元功能、阻碍神经系统再生等方式推动神经退行性疾病的形成和发展。衰老细胞清除剂是一类能选择性清除衰老细胞的化合物，能延长健康寿命，使个体在老龄化过程中减少慢性疾病困扰，包括减少肿瘤发生、延长中位生存期，减轻动脉粥样硬化、骨关节炎、白内障、黄斑变性、心肌肥大、慢性肾病、慢性肝病、肺纤维化、神经退行性疾病、脂肪代谢紊乱和肌肉减少症等老龄化改变。多个衰老细胞清除剂对衰老相关疾病治疗作用进入临床研究，如达沙替尼和槲皮素的组合物治疗特发性肺纤维化进入I期临床研究，UBX0101治疗骨关节炎进入II期临床(Ageing Research Reviews,2020,66:101251.)。衰老细胞在病变视网膜的微血管中蓄积，用药物局部清除衰老细胞可以有效阻止视网膜内病态血管新生，促进正常血管修复(Cell Metab,2021,33:818.)。衰老细胞清除剂UBX1325目前正在开展治疗糖尿病视网膜病变和年龄相关黄斑变性的临床试验。
除了一些亚群肿瘤细胞的OXPHOS增强以外，研究表明衰老细胞的OXPHOS过程同样增强。为了维持高水平的分泌表型，衰老细胞必须做出显著的代谢改变，比如耗氧量和能量生成增加，脂质分解更加活跃，同时产生高水平的ROS。相比于糖酵解，衰老细胞更加依赖于线粒体的OXPHOS作用提供能量，线粒体功能的增强是衰老细胞分泌SASP的一个重要决定因素。因此在理论上，靶向线粒体呼吸链复合物I、阻断OXPHOS是一种清除衰老细胞来的合理方法。有研究表明，线粒体靶向的他莫昔芬可以通过抑制线粒体呼吸链复合物I和降低线粒体膜电位而选择性地清除衰老细胞(Cell Death&Differentiation,2018,26(2),276-290.)。所以，应用线粒体呼吸链复合物I抑制剂来预防或治疗细胞衰老相关疾病或许是一种行之有效的方法。
文献报道(ACS Chemical Biology,2013,8(1),257–267.)，天然产物牛蒡子苷元(Arctigenin)是线粒体呼吸链复合物I的一个中等强度的选择性抑制剂。牛蒡子苷元具有抗炎、抗感染、抗代谢紊乱、抗神经损伤等药理作用，对于治疗癌症和动脉粥样硬化、糖尿病、阿尔兹海默症等衰老相关疾病具有很好的效果。但是它口服后易代谢，药代动力学性质差，生物利用率很低，限制了其作为口服药物的应用(Acta Pharmacologica Sinica,2018,39,787–801.)。
发明内容
本发明的目的在于提供一种对牛蒡子苷元进行结构改造所得的木脂素类衍生物，能够有效地抑制线粒体呼吸链复合物I。
本发明的第一方面，提供一种式I所示的化合物、其对映异构体、非对应异构体、外消旋混合物、氘代化物及药学上可接受的盐，
Figure PCTCN2022083003-appb-000001
其中，Rx选自氢、羟基、卤素；
R 0选自氢、卤素、C 1-C 6烷基、羟基取代的C 1-C 6烷基、C 1-C 6卤代烷基；
R 1选自卤素、C 1-C 6卤代烷基、-N(R 5) 2、C 1-C 6烷氧基、C 1-C 6氘代烷氧基或-O-L-G；其中，L为C 1-6亚烷基，所述亚烷基的氢原子任选地以化学键允许的方式和数量被卤素、羟基、＝O所取代；G为卤素、C 1-C 6卤代烷基、-CN、-N(R 9) 2、-COOR 10、-P +(R 11) 3Y -、-N +(R 11) 3Y -、C 6-10芳基、C 6-10芳氧基、4-8元杂环基或5-10元杂芳基；所述芳基、芳氧基、杂环基和杂芳基未被取代或被1、2、3或4个选自下组的基团所取代：卤素、羟基、-N(R 12) 2、硝基、氰基、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、4-8元杂环基；
R 2和R 3各自独立地为C 1-C 6烷基、C 1-C 6卤代烷基，或R 2和R 3连接起来并连同相连接的C形成未取代或取代的5-8元杂环基，所述取代是指被选自下组的一个或多个取代基取代：卤素、C 1-C 6烷基；
R 4为氢或C 1-C 6烷基；
各R 5各自独立地为氢、C 1-C 6烷基、-SO 2R 13、-COR 13；
Ry为羟基或
Figure PCTCN2022083003-appb-000002
各R 9各自独立地为氢、C 1-C 6烷基、C 6-10芳基、5-10元杂芳基，所述芳基、杂芳基可任选地被1-4个选自下组的基团所取代：卤素、羟基、-N(R 12) 2、硝基、氰基、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、4-8元杂环基；
各R 11各自独立地选自C 1-C 6烷基和C 6-10芳基，所述芳基任选地被1-4个选自下组的基团所取代：卤素、C 1-6烷基、C 1-6烷氧基；
R 8、R 10、R 12各自独立地选自氢、C 1-C 6烷基；
R 13为C 1-C 6烷基；
Y -为卤离子、甲酸根离子、乙酸根离子、三氟乙酸根离子或氢氧根离子；
m为0、1、2或3；各Q独立地为卤素或C 1-C 6烷基；
条件是当R 0、R 4、R x均为氢且Ry为羟基时，R 1不为C 1-C 6烷氧基。
本发明中，卤素包括氟、氯、溴、碘四个原子，卤离子为氟、氯、溴或碘的一价负离子。
在另一优选例中，R 1选自卤素、C 1-C 4卤代烷基、-N(R 5) 2、C 1-C 4烷氧基、C 1-C 4氘代烷 氧基或-O-L-G；其中，L为C 1-4亚烷基，所述亚烷基上的1、2或3个氢原子任选地以化学键允许的方式被卤素、羟基、＝O所取代；G为卤素、C 1-C 4卤代烷基、-CN、-N(R 9) 2、-COOR 10、-P +(R 11) 3Y -、-N +(R 11) 3Y -、苯基、苯基氧基、4-6元杂环基或6-8元杂芳基；所述苯基、苯基氧基、杂环基和杂芳基未被取代或被1、2或3个选自下组的基团所取代：卤素、羟基、-N(R 12) 2、硝基、氰基、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、4-8元杂环基。
在另一优选例中，各R 5各自独立地为氢或C 1-C 4烷基。在另一优选例中，各R 9各自独立地为氢或C 1-C 4烷基。在另一优选例中，R 10为氢或C 1-C 4烷基。在另一优选例中，各R 11各自独立地为C 1-C 4烷基或苯基。在另一优选例中，各R 12各自独立地为氢或C 1-C 4烷基。
在另一优选例中，Ry为羟基或
Figure PCTCN2022083003-appb-000003
各R 8各自独立地为氢或C 1-C 4烷基。
在另一优选例中，Rx为氢、氟、氯或溴。
在另一优选例中，R 0为氢、氟、氯、溴、C 1-C 4烷基、羟基取代的C 1-C 4烷基、C 1-C 4卤代烷基。
在另一优选例中，R 4为氢或C 1-C 4烷基。
在另一优选例中，R 2和R 3各自独立地为C 1-C 4烷基，或R 2和R 3连接起来并连同相连接的C形成
Figure PCTCN2022083003-appb-000004
R 6和R 7各自独立地为氢、C 1-C 4烷基或卤素。
在另一优选例中，所述化合物具有如下式(IIa)：
Figure PCTCN2022083003-appb-000005
其中，各取代基定义与前相同，当R 0为氢时，R 1不为C 1-C 6烷氧基。
在另一优选例中，所述化合物具有如下式(IIb)：
Figure PCTCN2022083003-appb-000006
其中，各取代基定义与权利要求1中相同。
在另一优选例中，各R 8为H。
在另一优选例中，R 1为C 1-C 4烷氧基或-O-L-G；其中，L为C 1-4亚烷基，所述亚烷基上的1或2个氢原子任选地以化学键允许的方式被氟、氯、溴、羟基、＝O所取代；G为氟、氯、溴、C 1-C 4卤代烷基、-CN、-N(R 9) 2、-COOR 10、-P +(R 11) 3Y -、-N +(R 11) 3Y -、苯基、苯基氧基、4-6元杂环基或6-8元杂芳基；所述苯基、苯基氧基、杂环基和杂芳基未被取代或被1、2或3个选自下组的基团所取代：卤素、羟基、-N(R 12) 2、硝基、氰基、C 1-C 4烷基、C 1-C 4卤代烷基、C 1-C 4烷氧基、4-8元杂环基；
其中，各R 5各自独立地为氢或C 1-C 4烷基；各R 9各自独立地为氢或C 1-C 4烷基；R 10为氢或C 1-C 4烷基；各R 11各自独立地为C 1-C 4烷基或苯基；各R 12各自独立地为氢或C 1-C 4烷基。
在另一优选例中，R 1为C 1-C 4烷氧基或-O-L-G；其中，L为C 1-4亚烷基；G为苯基或C 1-C 4卤代烷基；Ry为羟基或-OP(O)(OH) 2；R 0为氢或C 1-C 6烷基；R 2和R 3各自独立地为C 1-C 4烷基。
在另一优选例中，所述化合物选自：
Figure PCTCN2022083003-appb-000007
Figure PCTCN2022083003-appb-000008
Figure PCTCN2022083003-appb-000009
本发明的第二方面，提供一种药物组合物，所述药物组合物包含第一方面所述的化合物、 其对映异构体、非对应异构体、外消旋混合物、氘代化物及药学上可接受的盐；和
药学上可接受的载体。
本发明的第三方面，提供第一方面所述的化合物、其对映异构体、非对应异构体、外消旋混合物、氘代化物及药学上可接受的盐或者第二方面所述的药物组合物的用途，用于制备线粒体呼吸链复合体I抑制剂；用于制备预防和/或治疗与线粒体呼吸链复合物I活性或表达升高相关的疾病的药物；用于制备预防和/或治疗与线粒体氧化磷酸化作用增强相关的疾病的药物；用于制备预防和/或治疗与细胞衰老相关的疾病的药物；用于制备衰老细胞清除剂；或用于制备预防和/或治疗肿瘤的药物。
在另一优选例中，与线粒体氧化磷酸化作用增强相关的疾病为部分或完全依靠氧化磷酸化代谢途径供应能量的肿瘤。
在另一优选例中，与线粒体氧化磷酸化作用增强相关的疾病为细胞衰老相关的疾病。
在另一优选例中，所述肿瘤选自急性髓性白血病、神经胶质细胞瘤、淋巴瘤、胰腺癌、子宫癌、乳腺癌、非小细胞肺癌、肝细胞癌。
在另一优选例中，所述肿瘤为急性髓性白血病或神经胶质细胞瘤。
在另一优选例中，所述与细胞衰老相关的疾病选自：器官纤维化疾病、慢性肺病、慢性肾病、慢性肝病、骨关节炎、神经退行性疾病、炎症性肠病、动脉粥样硬化、青光眼、白内障、黄斑变性、糖尿病、糖尿病视网膜病变、色斑、少肌症。
在另一优选例中，所述与细胞衰老相关的疾病为器官纤维化疾病，选自肺纤维化、肝纤维化、肾纤维化。在另一优选例中，所述与细胞衰老相关的疾病为特发性肺纤维化。在另一优选例中，所述与细胞衰老相关的疾病为肝纤维化。在另一优选例中，所述与细胞衰老相关的疾病为骨关节炎。
在另一优选例中，所述与细胞衰老相关的疾病为神经退行性疾病，选自阿尔兹海默症、帕金森病、亨廷顿病、肌萎缩性侧索硬化、多发性硬化症。在另一优选例中，所述与细胞衰老相关的疾病为阿尔兹海默症。
在另一优选例中，所述与细胞衰老相关的疾病为炎症性肠病，选自溃疡性结肠炎、克罗恩病。
本发明还提供通式(I)所示化合物的制备方法，所述方法选自如下路线1或路线2，具体地，取决于五元内酯环的类型。
在下文中，保护基Pg是使得如下情况变得可能的基团：一方面在合成过程中保护反应性官能团，诸如羟基或胺，且在另一方面在合成结束时允许反应性官能团恢复完整。官能团的保护以及去保护的方法将在具体实施例中给出。
路线1适用于R 0选自氢、卤素、C 1-C 6烷基、羟基取代的C 1-C 6烷基、C 1-C 6卤代烷基；R 4选自氢；其他各取代基定义如前所述的化合物。
路线1：
Figure PCTCN2022083003-appb-000010
从购买或合成的苯丙酸衍生物1开始，与手性辅基4-苄基恶唑烷酮2缩合得到化合物3。随后化合物3在大位阻有机强碱的作用下发生羰基α位烷基化反应，得到化合物4。化合物4经还原剂还原消除手性辅基后得到构型单一的化合物5。化合物5在酸催化下发生酯交换反应得到关键中间体单一构型的丁内酯化合物6。随后再利用有机强碱拔氢、在化合物6的酯羰基α位引入取代苄基得到化合物8，化合物8的酚羟基脱保护得到通式(I)的化合物。化合物8再在有机强碱的作用下拔氢进攻亲电试剂得到α位取代的化合物9，化合物9的酚羟基脱保护得到通式(II)的化合物。
路线2适用于R 0选自氢，R 4选自C 1-C 6烷基，其他各取代基定义如前所述的化合物。
路线2：
Figure PCTCN2022083003-appb-000011
第二合成途径从中间化合物5开始，化合物5的羟基经氧化剂氧化得到相应的醛，随后格氏试剂与醛基反应游离出羟基，得到相应的化合物11。化合物11在酸催化下发生酯交换反应的到关键中间体丁内酯化合物12。随后再利用有机强碱拔氢在化合物12的酯羰基α位引入取代苄基化合物得到化合物13，化合物13的酚羟基脱保护得到通式(III)的化合物。
附图说明
图1示出小鼠在热板实验中的反应时间。
图2示出小鼠关节病理形态图片及OA评分。
图3示出关节软骨免疫组化p16阳性率。
图4示出IPF小鼠给药后体重变化情况。
图5示出小鼠给药后生存率变化情况。
图6示出小鼠肺组织切片Masson染色及病理评分。
图7示出小鼠肺组织衰老标记物p16基因水平。
图8示出小鼠肺组织SASP中IL-6基因水平。
图9示出在水迷宫实验中化合物A4-1a对转基因AD小鼠潜伏期的影响。
图10示出化合物A4-1a对转基因AD小鼠穿越平台数的影响。
图11示出化合物A5对四氯化碳诱导的肝纤维化小鼠肝组织内羟脯氨酸含量的影响。
图12示出化合物A5对肝纤维化小鼠肝脏Masson染色阳性率的影响。
图13示出化合物A5对恶唑酮诱导的肠炎小鼠疾病活动指数的影响。
图14示出化合物A5对恶唑酮诱导的肠炎小鼠结肠病理的影响。
图15示出化合物A4-1a和Arctigenin对小鼠口服30mg/kg后血浆、肝、结肠组织内药物浓度/时间变化曲线。
图16示出化合物A5对小鼠口服30mg/kg后血浆及组织内前药A5(A)和原形A4-1a(B)的药物浓度/时间变化曲线。
具体实施方式
本申请的发明人经过广泛而深入的研究，基于牛蒡子苷元进行结构改造，发现了线粒体呼吸链复合物I抑制活性提高、口服药代动力学性质改善、在动物模型上口服效果显著的一系列化合物，在用于制备药物时具有更好的成药性质和应用潜力。在此基础上，完成了本发明。
术语
在本发明中，除非特别指出，所用术语具有本领域技术人员公知的一般含义。
在本发明中，术语“C1-C6”是指具有1、2、3、4、5或6个碳原子，“C1-C8”是指具有1、2、3、4、5、6、7或8个碳原子，依此类推。“3-8元”是指具有3、4、5、6、7或8个环原子，“5-7元”是指具有5、6或7个环原子，依此类推。
本发明中，“芳环”、“芳基”定义为由特定个数碳原子构成、且遵守Hückel规则的单环和双环体系，包括但不仅限于苯环、萘环。
本发明中，“杂芳环”、“杂芳基”定义为具有特定个数成环原子、且含有1、2、3或4个杂原子(选自N、O、S)、同时遵守Hückel规则的单环和双环体系，包括但不仅限于吡啶、吡咯、咪唑。
本发明中，“芳氧基”定义为芳香性基团上连上氧形成的基团，包括但不限于Ph-O-。
本发明中，“杂环基”定义为由特定个数碳原子构成的饱和或不饱和的非芳族的环状基团，且含有1、2、3或4个杂原子(选自N、O、S)，包括但不仅限于吗啉、哌嗪。
本发明中，所述“烷基”和类似的“烷氧基”为具有特定碳原子个数的支链和直链的烃 基团和烃氧基团，代表性的例子包括但不仅限于甲基、甲氧基、乙基、乙氧基、正丙基、正丙氧基、异丙基、异丙氧基。
本发明中，所述“卤代烷基”是指具有特定碳原子个数的“烷基”中的氢原子被“卤原子”部分或完全取代所形成的基团。
本发明中，所述“卤原子”包括氟、氯、溴、碘。
本发明中，所述取代为单取代或多取代，所述多取代为二取代、三取代、四取代、或五取代。所述二取代就是指具有两个取代基，依此类推。
本发明所述药学上可接受的盐可以是阴离子与式I化合物上带正电荷的基团形成的盐。合适的阴离子为氯离子、溴离子、碘离子、硫酸根、硝酸根、磷酸根、柠檬酸根、甲基磺酸根、三氟乙酸根、乙酸根、苹果酸根、甲苯磺酸根、酒石酸根、富马酸根、谷氨酸根、葡糖醛酸根、乳酸根、戊二酸根或马来酸根。类似地，可以由阳离子与式I化合物上的带负电荷的基团形成盐。合适的阳离子包括钠离子、钾离子、镁离子、钙离子和铵离子，例如四甲基铵离子。在另一优选例中，“药学上可接受的盐”是指式I化合物同选自下组的酸形成的盐类：氢氟酸、盐酸、氢溴酸、磷酸、乙酸、草酸、硫酸、硝酸、甲磺酸、胺基磺酸、水杨酸、三氟甲磺酸、萘磺酸、马来酸、柠檬酸、醋酸、乳酸、酒石酸、琥珀酸、酢浆草酸、丙酮酸、苹果酸、谷氨酸、对甲苯磺酸、萘磺酸、乙磺酸、萘二磺酸、丙二酸、富马酸、丙酸、草酸、三氟乙酸、硬酯酸、扑酸、羟基马来酸、苯乙酸、苯甲酸、谷氨酸、抗坏血酸、对胺基苯磺酸、2-乙酰氧基苯甲酸和羟乙磺酸等；或者式I化合物与无机碱形成的钠盐、钾盐、钙盐、铝盐或铵盐；或者通式I化合物与有机碱形成的甲胺盐、乙胺盐或乙醇胺盐。
本发明通式I化合物或其药学上可接受的盐是从水或有机溶剂中蒸馏析出、结晶或重结晶而来，化合物中可能包含所使用的溶剂分子。此外，不同的结晶条件可能导致化合物的晶型不同。因此，含有不同化学剂量的结晶溶剂以及所有晶型的通式I所示的化合物或其药学上可接受的盐都在本发明的范围内。本发明中，通式(I)所示化合物用于制备药物时通常为药物组合物的形式，该药物组合物包含通式(I)化合物以及一种或多种药学上可接受的辅料；所述药学上可接受的辅料为药学上可接受的载体、赋形剂、缓释剂、气味剂、香味剂等；所述药物组合物中，通式(I)所示化合物作为活性组分、其重量占药物组合物总重量的0.1～99.9％，其余为药学上可接受的辅料；所述药物组合物可以基于药物制剂领域的惯用工艺制成多种剂型，如片剂、胶囊、溶液、混悬液、气雾剂、干粉等，并可存储于适宜的消毒器具和给药装置中。
本发明中，所述“有效治疗剂量”表示与没有接受该剂量治疗的对象相比，接受该剂量治疗的对象在病变或副作用等得到治愈、改善、有效预防或者其发生率显著降低；此外，它还包括增强正常生理功能的有效剂量。
本发明中，通式(I)所示化合物、其盐及其药物组合物可对人和动物使用；给药途径包括口服、吸入、透皮吸收、注射等；本发明中，优选的给药途径为口服；通式(I)所示化合物、其盐及其药物组合物用于制备药物时；给药剂量和频率应根据医嘱而定。
本发明中，通式(I)所示化合物、其盐及其药物组合物用于预防或治疗癌症和衰老相关疾 病时可与其他药物联合使用；所述药物包括但不仅限于抗肿瘤药物、治疗组织纤维化药物、治疗骨关节炎药物。
本发明中，所述“酸”、“碱”、“氧化剂”、“还原剂”均与本领域熟练人员所熟悉的意义相同，本领域熟练技术人员可根据具体反应底物、反应条件的不同进行适当地变化和调整。
以下将以实施例进一步说明本发明。需要特别指出的是，这些实施例只用于举例说明本发明，而不以任何方式限制本发明。实例中的所有参数及其余说明，除另加说明外，都是以质量为依据的。柱层析分离所用填料若未说明均为硅胶。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
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中。文中所述的较佳实施方法与材料仅作示范之用。
缩略语
Bn：苄基；DCM：二氯甲烷；DIPEA：N,N-二异丙基乙胺；DMAP：4-二甲氨基吡啶；DMF：N,N-二甲基甲酰胺；THF：四氢呋喃；LDA：二异丙基氨基锂；NaHMDS：双(三甲基硅基)氨基钠。
实施例1：(3R,4R)-4-(3,4-二甲氧基苄基)-3-(4-羟基-3-甲氧基苄基)-3-甲基二氢呋喃-2(3H)-酮(A4-1a)
Figure PCTCN2022083003-appb-000012
(3R,4R)-3-(4-(苄氧基)-3-甲氧基苄基)-4-(3,4-二甲氧基苄基)二氢呋喃-2(3H)-酮(A2-1)的合成
将市售的原料牛蒡子苷元A1-1(3.72g,10.0mmol)溶于60ml丙酮中，再将氯化苄(2.3mL,20mmol)，碳酸钾(2.76g,20mmol)，碘化钾(63mg,0.5mmol)加入反应体系中，60℃油浴加热搅拌6h.反应完全后，抽滤,滤液减压浓缩,硅胶柱层析[PE:EA＝1∶1],得4.32g透明油状物A2-1,产率93.3％。MS m/z:463.2[M+H] +
(3R,4R)-3-(4-(苄氧基)-3-甲氧基苄基)-4-(3,4-二甲氧基苄基)-3-甲基二氢呋喃-2(3H)-酮(A3-1)的合成
将上一步产物A2-1(463mg，1mmol)溶于20ml无水THF中，于-78℃滴加LDA(1mL,2mmol,2mol/L in THF).搅拌40min滴加碘甲烷(186μL,3mmol in 5ml THF)，于-78℃下搅拌30min后缓慢升温到室温。反应完全后饱和氯化铵溶液淬灭反应,减压浓缩除去大部分四氢呋喃,剩余物溶于20mL乙酸乙酯,氯化钠溶液洗涤,无水硫酸钠干燥。抽滤,滤液减压浓缩,硅胶柱层析[PE:EA＝1∶1]，得278mg透明油状物A3-1，产率58.4％。 1H NMR(500MHz,Chloroform-d)δ7.46–7.41(m,2H),7.38–7.33(m,2H),7.31–7.27(m,1H),6.81(d,J＝2.0Hz, 2H),6.77(d,J＝2.1Hz,1H),6.71(dd,J＝8.2,2.1Hz,1H),6.67(dd,J＝8.1,2.0Hz,1H),6.63(d,J＝2.0Hz,1H),5.13(s,2H),4.02(dd,J＝9.0,7.5Hz,1H),3.88(s,3H),3.87(s,3H),3.86(s,3H),3.62(dd,J＝10.2,9.0Hz,1H),2.96(dd,J＝13.6,4.8Hz,1H),2.87(d,J＝14.0Hz,1H),2.79(d,J＝14.1Hz,1H),2.57(m,1H),1.23(s,3H).ESI-MS m/z:477.2[M+H] +。
(3R,4R)-4-(3,4-二甲氧基苄基)-3-(4-羟基-3-甲氧基苄基)-3-甲基二氢呋喃-2(3H)-酮(A4-1a)的合成
将上一步产物A3-1(278mg，0.58mmol)溶于10ml无水甲醇中，加入10％钯碳(30mg)，压力为1大气压加氢反应2h完毕。过滤除去催化剂，滤液减压浓缩，硅胶柱层析[PE:EA＝1∶1]分离得189mg白色固体A4-1a，产率84％。 1H NMR(500MHz,Chloroform-d)δ6.84(d,J＝8.0Hz,1H),6.76(d,J＝8.2Hz,1H),6.74(d,J＝2.1Hz,1H),6.64(dd,J＝8.0,2.0Hz,1H),6.60(dd,J＝8.1,2.0Hz,1H),6.52(d,J＝1.9Hz,1H),3.94(dd,J＝9.0,7.8Hz,1H),3.84(s,3H),3.84(s,4H),3.83(s,4H),3.82(d,J＝1.3Hz,1H),3.19(d,J＝14.0Hz,1H),2.73(dd,J＝13.5,4.0Hz,1H),2.62(d,J＝14.0Hz,1H),2.58–2.45(m,1H),2.36(dd,J＝13.6,11.1Hz,1H),1.28(s,3H).ESI-MS m/z:387.2[M+H] +。
采用本发明实施例1的类似合成方法，以合适的可选起始原料，制备得到表1所示的化合物：
表1 化合物及其表征数据
Figure PCTCN2022083003-appb-000013
Figure PCTCN2022083003-appb-000014
Figure PCTCN2022083003-appb-000015
实施例2：(3R,4R)-4-(3,4-二甲氧基苄基)-3-(4-羟基-3-苯乙氧基苄基)二氢呋喃-2(3H)-酮(B9-1a)
Figure PCTCN2022083003-appb-000016
(S)-4-苄基-3-(3-(3,4-二甲氧基苯基)丙酰基)-2-噁唑烷酮(B2-1)
将3,4-二甲氧基苯丙酸(B1-1)(10.50g,50.0mmol)溶于300mL无水THF中,于－20℃滴加特戊酰氯(6.0mL,50.0mmol)和三乙胺(21.0mL,150.0mmol),此温度下继续搅拌1h.滴加(S)-4-苄基-2-噁唑烷酮(B1-2)(8.0g,45.0mmol)的THF溶液(100mL),一次性加入无水LiCl(2.10g,50.0mmol),继续搅拌1h后升至室温,再继续搅拌4h.反应液减压浓缩至约50mL,以100mL 乙酸乙酯稀释,依次用10％碳酸氢钠、5％硫酸氢钾、饱和氯化钠溶液洗涤,无水硫酸钠干燥。抽滤,滤液减压浓缩,得15.8g白色固体B2-1,不经纯化,可直接用于下步反应。 1H NMR(400MHz,Chloroform-d)δ7.35–7.27(m,3H),7.20–7.13(m,2H),6.81(dd,J＝3.4,1.7Hz,3H),4.66(ddt,J＝9.4,6.9,3.5Hz,1H),4.22–4.03(m,2H),3.88(s,3H),3.85(s,3H),3.36–3.17(m,3H),3.05–2.89(m,2H),2.75(dd,J＝13.4,9.5Hz,1H).ESI-MS m/z:370.2[M+H] +。
(S)-4-苄基-3-[(R)-2-乙酸叔丁酯基-3-(3,4-二甲氧基苯基)丙酰基]-2-噁唑烷酮(B3-1)
将上步产物B2-1(15.8g,43mmol)溶于500mL无水THF,于-78℃滴加NaHMDS(32mL,64mmol,2mol/L in THF).搅拌1小时滴加溴乙酸叔丁酯(12.6mL,85mmol),继续在此温度下搅拌5h.饱和氯化铵溶液淬灭反应,减压浓缩除去大部分四氢呋喃,剩余物溶于100mL乙酸乙酯,依次用5％硫酸氢钾和饱和氯化钠溶液洗涤,无水硫酸钠干燥.抽滤,滤液减压浓缩,硅胶柱层析[PE:EA＝4∶1],得28.6g白色固体B3-1,两步产率59.2％. 1H NMR(400MHz,Chloroform-d)δ7.35–7.31(m,2H),7.27(s,3H),6.87(s,1H),6.76(d,J＝1.5Hz,2H),4.60–4.50(m,1H),4.45(ddd,J＝10.1,8.4,5.0Hz,1H),4.12(d,J＝7.1Hz,1H),4.10–4.06(m,1H),3.97(t,J＝8.4Hz,1H),3.89(s,3H),3.85(s,3H),3.35–3.26(m,1H),2.96(dd,J＝13.1,6.0Hz,1H),2.85–2.75(m,1H),2.78–2.70(m,1H),2.56(dd,J＝13.2,9.3Hz,1H),2.38(dd,J＝16.9,4.0Hz,1H),1.46(d,J＝1.8Hz,1H),1.40(s,9H).ESI-MS m/z:484.2[M+H] +.
(R)-3-羟甲基-4-(3,4-二甲氧基苯基)-丁酸叔丁酯(B4-1)
将上一步产物B3-1(2.50g,5.2mmol)溶于80mL四氢呋喃中,加入20mL水,分批加入硼氢化钠(0.29g,5.8mmol),室温搅拌2h.减压浓缩除去大部分四氢呋喃,乙酸乙酯稀释,缓慢滴加1mol/L盐酸至无气泡产生.依次用5％硫酸氢钾和饱和氯化钠洗涤,无水硫酸钠干燥.抽滤,滤液减压浓缩,得1.52g无色透明液体B4-1.不经纯化,可直接用于下步反应. 1H NMR(400MHz,Chloroform-d)δ6.83–6.69(m,3H),3.87(s,3H),3.85(s,3H),2.65(dd,J＝13.6,6.1Hz,1H),2.55(dd,J＝14.0,5.9Hz,1H),2.35–2.19(m,3H),2.01(dd,J＝14.3,8.7Hz,1H),1.45(s,9H).ESI-MS m/z:311.2[M+H] +
(R)-4-(3,4-二甲氧基苄基)-丁内酯(B5-1)
将上步产物B4-1(1.50g,4.8mmol)溶于20mL甲苯,加入对甲苯磺酸(41.6mg,0.24mmol),于80℃搅拌1小时.减压浓缩除去甲苯.残渣溶于乙酸乙酯,依次用10％碳酸氢钠和饱和氯化钠溶液洗涤,无水硫酸钠干燥,硅胶柱层析[V(石油醚)∶V(乙酸乙酯)＝3∶1],得1.14g无色透明油状物B5-1。 1H NMR(400MHz,Chloroform-d)δ6.81(d,J＝8.1Hz,1H),6.70(dd,J＝8.1,2.0Hz,1H),6.66(d,J＝2.0Hz,1H),4.34(dd,J＝9.1,6.9Hz,1H),4.04(dd,J＝9.1,6.0Hz,1H),3.88(s,3H),3.87(s,3H),2.89–2.78(m,1H),2.72(dd,J＝7.7,4.1Hz,2H),2.61(dd,J＝17.5,8.1Hz,1H),2.29(dd,J＝17.5,6.9Hz,1H).ESI-MS m/z:237.1[M+H] +
4-(苄氧基)-3-羟基苯甲醛(B5-2b)的合成
将市售原料B5-2a(1.38g,10mmol)溶于20ml丙酮中，加入碳酸锂(888mg，12mmol)，氯化苄(1.37ml，12mmol)，碘化钾(83mg，0.5mmol)，60℃油浴加热搅拌6h，反应完毕后抽滤除去碳酸锂，滤液减压蒸馏，硅胶柱层析[PE:EA＝3∶1]分离得1.82g白色固体B5-2b，产率79.8％。 1H NMR(500MHz,Chloroform-d)δ9.84(s,1H),7.46(d,J＝2.0Hz,1H),7.43(m,4H),7.41–7.38(m,2H),7.04(d,J＝8.2Hz,1H),5.21(s,2H).MS m/z:229.1[M+H] +
4-(苄氧基)-3-((叔丁基二甲基甲硅烷基)氧基)苯甲醛(B5-2c)的合成
上一步产物B5-2b(1.14g，5mmol)溶于20ml二氯甲烷中，冰浴下加N,N-二异丙基乙胺(2.6ml，15mmol)，叔丁基二甲基氯硅烷(1.5g，10mmol)，冰浴搅拌30min后，恢复至室温搅拌。反应完毕后，减压浓缩反应液，硅胶柱层析[PE:EA＝5∶1]分离得1.56g白色固体B5-2c，产率91.2％。 1H NMR(500MHz,Chloroform-d)δ9.70(s,1H),7.30-7.34(m,3H),7.29–7.20(m,4H),6.90(d,J＝8.3Hz,1H),5.01(s,2H),0.84(s,9H).MS m/z:343.2[M+H] +
(4-(苄氧基)-3-((叔丁基二甲基甲硅烷基)氧基)苯基)甲醇(B5-2d)的合成
上一步产物B5-2c(1.03g,3mmol)溶于10ml四氢呋喃中，温室分批加入硼氢化钠(228mg，6mmol)，温室搅拌3h,反应完毕后，硅胶柱层析[PE:EA＝3∶1]分离得986mg白色固体B5-2d，产率95.5％。 1H NMR(600MHz,Chloroform-d)δ7.35–7.29(m,2H),7.28–7.23(m,2H),7.22–7.19(m,1H),6.78(d,J＝1.5Hz,1H),6.76(d,J＝1.9Hz,2H),4.93(s,2H),4.44(s,2H),0.85(s,9H).MS m/z:345.2[M+H] +
(2-(苄氧基)-5-(溴甲基)苯氧基)(叔丁基)二甲基硅烷(B5-2e)的合成
三苯基膦(688mg,3mmol)和四溴化碳(996mg,3mmol)溶于5ml二氯甲烷中，室温搅拌5min后，向反应体系中滴加B5-2d(688mg,2mmol)的二氯甲烷(5ml)溶液，温室搅拌4h,反应完毕后，减压浓缩反应液，硅胶柱层析[PE:EA＝5∶1]分离得952mg油状物B5-2e，产率78.1％。 1H NMR(500MHz,Chloroform-d)δ7.47–7.44(m,2H),7.43–7.38(m,2H),7.38–7.34(m,1H),6.94(d,J＝1.4Hz,1H),6.92(d,J＝2.2Hz,1H),6.90–6.87(m,1H),5.08(s,2H),4.54(s,2H),1.00(s,9H).MS m/z:407.1[M+H] +
(3R,4R)-3-(4-(苄氧基)-3-((叔丁基二甲基甲硅烷基)氧基)苄基)-4-(3,4-二甲氧基苄基)二氢呋喃-2(3H)-酮(B6-1)的合成
将上步产物B5-1(400.0mg,1.7mmol)的10mL四氢呋喃溶液,于－78℃滴加LDA(1.7mL,3.4mmol,2mol/L in THF).搅拌40min滴加滴加化合物B5-2e(690.2mg,1.7mmol)的5mL四氢呋喃溶液,继续搅拌4h,饱和氯化铵淬灭反应,减压浓缩除去大部分四氢呋喃,剩余物溶于乙酸乙酯,依次用5％硫酸氢钾和饱和氯化钠洗涤,无水硫酸钠干燥后,硅胶柱层析[V(石油醚)∶V(乙酸乙酯)＝3∶1],得614mg无色粘稠液体B6-1,收率64.3％.MS m/z:563.3[M+H] +
(3R,4R)-3-(4-(苄氧基)-3-羟基苄基)-4-(3,4-二甲氧基苄基)二氢呋喃-2(3H)-酮(B7-1)的合成
将上步产物B6-1(300.0mg,0.53mmol)溶于5ml盐酸/乙醇(2mol/L)溶液中,于室温下搅拌2h，反应完毕后减压蒸馏除去大部分溶剂,硅胶柱层析[V(石油醚)∶V(乙酸乙酯)＝3∶1],得165mg无色粘稠液体B7-1,收率73.6％.MS m/z:449.2[M+H] +
(3R,4R)-3-(4-(苄氧基)-3-苯乙氧基苄基)-4-(3,4-二甲氧基苄基)二氢呋喃-2(3H)-酮(B8-1a)的合成
将上步产物B7-1(165mg,0.37mmol)溶于5mL乙腈中,室温下加碳酸铯(240mg,0.74mmol)，市售化合物B7-2a(272mg，1.48mmol)。80℃油浴搅拌2h，反应完毕后抽滤除去碳酸铯,滤液减压蒸馏，硅胶柱层析[V(石油醚)∶V(乙酸乙酯)＝3∶1],得195mg油状物B8-1a,收率95.5％.MS m/z:553.2[M+H] +
(3R,4R)-4-(3,4-二甲氧基苄基)-3-(4-羟基-3-苯乙氧基苄基)二氢呋喃-2(3H)-酮(B9-1a)的合成
将上一步产物B8-1a(195mg，0.35mmol)溶于10ml无水甲醇中，加入10％钯碳(20mg)，压力为1大气压加氢反应2h完毕。过滤除去催化剂，滤液减压浓缩，硅胶柱层析[PE:EA＝2∶1]分离得136mg白色固体B9-1a，产率84.1％。 1H NMR(500MHz,Chloroform-d)δ7.33(t,J＝7.6Hz,2H),7.29–7.24(m,3H),6.81(dd,J＝8.3,2.8Hz,1H),6.73(dd,J＝9.5,4.0Hz,1H),6.67(d,J＝1.9Hz,1H),6.60(dd,J＝8.1,2.0Hz,1H),6.54(dd,J＝8.1,2.0Hz,1H),6.47(d,J＝2.0Hz,1H),4.22–4.17(m,2H),4.10(dd,J＝8.9,7.0Hz,1H),3.87(dd,J＝7.5,8.4Hz,1H),3.84(s,3H),3.80(s,3H),3.10(q,J＝7.6,6.8Hz,2H),2.90(t,J＝4.2Hz,2H),2.64(dd,J＝13.1,5.6Hz,1H),2.59–2.42(m,2H).MS m/z:463.2[M+H] +
采用实施例2类似的合成方法，以合适的可选不同的起始原料，制备得到表2所示的化合物：
表2 化合物及其表征数据
Figure PCTCN2022083003-appb-000017
Figure PCTCN2022083003-appb-000018
Figure PCTCN2022083003-appb-000019
Figure PCTCN2022083003-appb-000020
实施例3:(3R,4R)-4-(3,4-二甲氧基苄基)-3-(4-羟基-3-苯乙氧基苄基)-3-甲基二氢呋喃-2(3H)-酮(B11-1a)
Figure PCTCN2022083003-appb-000021
(3R,4R)-3-(4-(苄氧基)-3-苯乙氧基苄基)-4-(3,4-二甲氧基苄基)-3-甲基二氢呋喃-2(3H)-酮(B10-1a)的合成
以化合物B8-1a为原料，合成方法参照化合物A3-1合成。ESI-MS m/z:567.3[M+H] +
(3R,4R)-4-(3,4-二甲氧基苄基)-3-(4-羟基-3-苯乙氧基苄基)-3-甲基二氢呋喃-2(3H)-酮(B11-1a)的合成
以化合物B10-1a为原料，合成方法参照化合物A4-1a的合成。 1H NMR(500MHz,Chloroform-d)δ7.34–7.29(m,2H),7.29–7.26(m,3H),6.82(d,J＝8.1Hz,1H),6.76(d,J＝1.9Hz,1H),6.74–6.71(m,1H),6.66–6.62(m,1H),6.59(dd,J＝8.1,2.0Hz,1H),6.51(d,J＝2.0Hz,1H),4.25(dd,J＝9.0,7.7Hz,1H),4.21(td,J＝6.8,1.5Hz,2H),3.94(dd,J＝9.0,7.7Hz,1H),,3.83(s,3H),3.82(s,3H),3.17(d,J＝14.0Hz,1H),3.09(t,J＝6.8Hz,2H),2.72(dd,J＝13.5,4.1Hz,1H),2.60(d,J＝14.0Hz,1H),2.48-2.54(m,1H),2.40–2.31(m,1H),1.27(s,3H).ESI-MS m/z:477.2[M+H] +
采用实施例3类似的合成方法，以合适的可选起始原料，制备得到表3所示的化合物：
表3 化合物及其表征数据
Figure PCTCN2022083003-appb-000022
Figure PCTCN2022083003-appb-000023
Figure PCTCN2022083003-appb-000024
实施例4：(3R,4R)-4-(3,4-二甲氧基苄基)-3-(4-羟基-3-(甲基氨基)苄基)二氢呋喃-2(3H)-酮(B9-3)
Figure PCTCN2022083003-appb-000025
4-(苄氧基)-3-硝基苯甲酸甲酯(B5-3b)的合成
市售原料B5-3a(1g，5mmol)，氯化苄(687ul,6mmol),碳酸钾(1.38g，10mmol)，碘化钾(83mg，0.5mmol)加入30ml丙酮中，60℃加热搅拌5小时。反应完毕后，抽滤除去碳酸钾，滤液减压蒸馏。硅胶柱层析[V(石油醚)∶V(乙酸乙酯)＝3∶1],得1.32淡黄色固体B5-3b,收率91.9％。 1H NMR(500MHz,Chloroform-d)δ8.52(d,J＝2.1Hz,1H),8.17(dd,J＝8.8,2.2Hz,1H),7.49–7.32(m,5H),7.16(d,J＝8.8Hz,1H),5.31(s,2H),3.93(s,3H).ESI-MS m/z:288.1[M+H] +
(4-(苄氧基)-3-硝基苯基)甲醇(B5-3c)的合成
上一步产物B5-3b(1.04g，4mmol)溶于20mL的无水THF中，冰浴下滴加四氢铝锂溶液(2.5M/L in THF)(2.5ml,10mmol),冰浴搅拌30分钟后缓慢升温到室温反应2小时。反应完毕后冰水淬灭四氢铝锂，乙酸乙酯萃取三次，饱和氯化钠溶液水洗有机层三次，无水硫酸钠干燥有机层，抽滤，滤液减压浓缩，硅胶柱层析[V(石油醚)∶V(乙酸乙酯)＝2∶1],得864mg黄色油状物B5-3c,收率83.1％。 1H NMR(500MHz,Chloroform-d)δ7.89(d,J＝2.2Hz,1H),7.52(dd,J＝8.6,2.3Hz,1H),7.50–7.46(m,2H),7.45–7.40(m,2H),7.38–7.33(m,1H),7.13(d,J＝8.6Hz,1H),5.27(s,2H),4.71(s,2H).ESI-MS m/z:260.1[M+H] +
1-(苄氧基)-4-(溴甲基)-2-硝基苯(B5-3e)
以B5-3c为原料，参照B5-2e的操作，合成得黄色油状物B5-3e，收率86％。ESI-MS m/z:322.0[M+H] +
(3R,4R)-3-(4-(苄氧基)-3-硝基苄基)-4-(3,4-二甲氧基苄基)二氢呋喃-2(3H)-酮(B6-3)的合成
以B5-3e和B5-1为原料，参照B6-1的操作，合成得淡黄色油状物，收率78％。ESI-MS m/z:478.2[M+H] +
(3R,4R)-3-(3-氨基-4-(苄氧基)苄基)-4-(3,4-二甲氧基苄基)二氢呋喃-2(3H)-酮(B7-3)的合成
上一步产物B6-3(477mg，1mmol)，铁粉(280mg，5mmol)溶于10ml甲醇中，加1ml稀盐酸(2M/L)催化，室温搅拌，反应过夜，反应完毕后过滤除去铁粉，滤液减压浓缩，硅胶柱层析[V(石油醚)∶V(乙酸乙酯)＝1∶1],得421g淡黄色油状物B7-3,收率94.2％。ESI-MS m/z:448.2[M+H] +
(3R,4R)-3-(4-(苄氧基)-3-(甲基氨基)苄基)-4-(3,4-二甲氧基苄基)二氢呋喃-2(3H)-酮(B8-3)的合成
上一步产物B7-3(223mg，0.5mmol)，N,N-二异丙基乙胺(350ul,2mmol)，碘甲烷(40ul，0.6mmol)加入5ml二氯甲烷中，室温搅拌5h，反应完毕后，硅胶柱层析[V(石油醚)∶V(乙酸乙酯)＝1∶1],得167mg淡黄色油状物B8-3,收率72.6％。ESI-MS m/z:462.2[M+H] +
(3R,4R)-4-(3,4-二甲氧基苄基)-3-(4-羟基-3-(甲基氨基)苄基)二氢呋喃-2(3H)-酮(B9-3)的合成
以B8-3为原料，参照合成A4-1a的操作步骤以及投料比，得到B9-3，收率78.2％。 1H NMR(500MHz,Chloroform-d)δ7.10(d,J＝8.1Hz,1H),6.79(d,J＝8.1Hz,1H),6.59(dd,J＝8.1,2.0Hz,1H),6.50(ddd,J＝12.0,8.0,2.1Hz,3H),4.18(dd,J＝9.2,7.5Hz,1H),3.92–3.87(m,1H),3.87(s,4H),3.83(s,3H),3.17(s,3H),2.88(dd,J＝6.0,3.7Hz,2H),2.66(dd,J＝13.8,6.7Hz,1H),2.62–2.55(m,2H),2.51–2.43(m,1H).ESI-MS m/z:372.2[M+H] +
采用实施例4类似的合成方法，以合适的可选起始原料，制备得到表4所示的化合物。
表4 化合物及其表征数据
Figure PCTCN2022083003-appb-000026
Figure PCTCN2022083003-appb-000027
实施例5：(3R,4R)-4-(3,4-二甲氧基苄基)-3-(4-羟基-3-甲氧基苄基)-5-甲基二氢呋喃-2(3H)-酮(C9-1a)
Figure PCTCN2022083003-appb-000028
3-(3,4-二甲氧基苄基)-4-氧代丁酸叔丁酯(C5-1)的合成
将中间体B4-1(620mg，2mmol)溶于10mL的二氯甲烷中，加入戴斯马丁试剂(932mg，2.2mmol)，室温搅拌1小时，反应完成后，硫代硫酸钠水溶液淬灭反应，二氯甲烷萃取3次，水洗有机层3次，无水硫酸钠干燥有机层，抽滤，滤液减压浓缩，硅胶柱层析[V(石油醚):V(乙酸乙酯)＝4∶1],得587mg白色油状物C5-1，收率96.5％。ESI-MS m/z:309.2[M+H] +。
3-(3,4-二甲氧基苄基)-4-羟基戊酸叔丁酯(C6-1a)的合成
将中间体C5-1(587mg，1.9mmol)溶于超干四氢呋喃中，于冰浴下滴加甲基溴化镁溶液(1M/L in THF)(2mL,2mmol),冰浴搅拌30分钟后缓慢升温到室温反应2小时。反应完毕后冰水淬灭甲基溴化镁溶液，乙酸乙酯萃取三次，饱和氯化钠溶液水洗有机层三次，无水硫酸钠干燥有机层，抽滤，滤液减压浓缩，硅胶柱层析[V(石油醚)∶V(乙酸乙酯)＝4∶1],得592mg透明油状物C6-1a,收率91.1％。ESI-MS m/z:325.2[M+H] +。
4-(3,4-二甲氧基苄基)-5-甲基二氢呋喃-2(3H)-酮(C7-1a)的合成
以C6-1a为原料，参照合成B5-1的操作步骤以及投料比，得到C7-1a，收率82.2％。ESI-MS m/z:251.1[M+H] +。
3-(4-(苄氧基)-3-甲氧基苄基)-4-(3,4-二甲氧基苄基)-5-甲基二氢呋喃-2(3H)-酮(C8-1)
以C7-1a和7为原料，参照合成B6-1的操作步骤以及投料比，得到C8-1，收率56.3％。ESI-MS m/z:477.2[M+H] +。
4-(3,4-二甲氧基苄基)-3-(4-羟基-3-甲氧基苄基)-5-甲基二氢呋喃-2(3H)-酮(C9-1a)
以C8-1为原料，参照合成A4-1a的操作步骤以及投料比，得到C9-1a，收率87.4％。1H NMR(400MHz,Chloroform-d)δ6.77(d,J＝8.0Hz,1H),6.71(d,J＝8.1Hz,1H),6.57(d,J＝1.9Hz,0H),6.51(dt,J＝7.9,1.8Hz,1H),6.48(d,J＝1.9Hz,1H),6.41(d,J＝2.0Hz,1H),3.86(d,J＝3.4Hz,3H),3.83(d,J＝4.1Hz,2H),3.78–3.69(m,5H),2.96(dd,J＝14.2,5.9Hz,0H),2.90–2.82(m,1H),2.70–2.59(m,2H),2.61–2.46(m,1H),1.33(d,J＝6.6Hz,2H),1.07(d,J＝6.2Hz,1H).ESI-MS m/z:387.2[M+H] +。
采用实施例5类似的合成方法，以合适的可选起始原料，制备得到表5所示的化合物。
表5 化合物及其表征数据
Figure PCTCN2022083003-appb-000029
实施例6：4-(((3R,4R)-4-(3,4-二甲氧基苄基)-3-甲基-2-氧代四氢呋喃-3-基)甲基)-2-甲氧基苯基磷酸二氢酯(A5)
Figure PCTCN2022083003-appb-000030
将化合物A4-1a(0.1mmol)溶于超干二氯甲烷中，冰浴下加入三乙胺(0.5mmol)，搅拌均匀后加入三氯氧磷(0.2mmol)，冰浴下反应30分钟。冰水淬灭反应，二氯甲烷萃取反应液，无水硫酸镁干燥，过滤，减压蒸馏，半制备液相纯化得化合物A5。 1H NMR(500MHz,DMSO-d6)δ7.21(d,J＝8.2Hz,1H),6.92(d,J＝2.0Hz,1H),6.86(d,J＝8.2Hz,1H),6.75(dd,J＝8.3,2.0Hz,1H),6.71(d,J＝2.0Hz,1H),6.67(dd,J＝8.0,2.1Hz,1H),4.02(t,J＝9.4Hz,1H),3.94(t,J＝8.1Hz,1H),3.74(s,3H),3.72(s,3H),3.71(s,3H),2.99(d,J＝13.6Hz,1H),2.75(d,J＝13.6Hz,1H),2.69–2.62(m,1H),2.48–2.37(m,1H),2.00(dt,J＝15.9,6.9Hz,2H),1.22(s,3H).MS(ESI,m/z):465.2[M-1] -。
采用实施例6类似的合成方法，以合适的可选起始原料，制备得到表6所示的化合物。
Figure PCTCN2022083003-appb-000031
实施例7药理活性和药效评价
1、化合物对线粒体呼吸复合物I(Complex I)抑制活性评价
1.1评价方法
线粒体提取于小鼠心脏组织。心脏组织加入200-250mL的solution A(0.22M mannitol(甘露醇)，0.07M sucrose(蔗糖)，0.02M HEPES，2mM Tris-HCl，1mM EDTA，pH 7.2，0.4％BSA)，组织剪碎，然后用solution A再清洗3次去除血液和结缔组织。采用玻璃匀浆器，对组织进行 匀浆，30次左右。悬液于3000g 1.5分钟，取上清留用，沉淀solution A加入蛋白酶抑制剂再次重悬后重新离心，收集上清，两次上清混合进行下一步。上清于17500g离心2.5分钟，弃上清，沉淀采用solution A加入蛋白酶抑制剂再次重悬后，再次17500g离心4.5分钟。Solution B(2.5mM potassium phosphate，5mM MgCl 2pH 7.2)再次重悬后进行反复冻融制得后续用于检测的线粒体复合物溶液。
Complex I的NADH活性测定，2.5mL反应液配制：50mM HEPES，5mM MgCl 2pH 7.5加入30μg上述过程中的线粒体蛋白。然后加入需要检测的化合物平衡5分钟，随后分装至每一个孔，最后快速加入NADH(50μM),于340nM每30sec读数一次，一共进行15分钟反应。计算：不同化合物中NADH反应速率计算，然后计算抑制率(化合物反应速率/对照反应速率*100)，最后再计算化合物的IC 50值。
1.2评价结果
表6 化合物对Complex I的抑制活性
化合物 Complex I IC 50(nM) 化合物 Complex I IC 50(nM)
A4-1a 507 B9-1u 564
A4-1b 1271 B9-1v 34
A4-1c 185 B9-1x 650
A4-1e 473 B11-1a 646
B9-1a 54 B11-1b 668
B9-1d 696 B11-1e 1374
B9-1p 465 B11-1f 420
B9-1q 64 B11-1h 13220
B9-1r 316    
由评价结果可见，本发明化合物具有微摩尔至纳摩尔水平的Complex I抑制活性，能用于制备治疗线粒体呼吸链复合物I功能异常所致疾病的药物。。
2、化合物对人急性髓细胞白血病细胞(OCI-AML3)活力影响的评价
2.1实验方法
OCI-AML3细胞为悬浮细胞，培养于RPIM1640+10％FBS+1％PS培养液中，37℃，5％CO 2。OCI-AML3按照20000个细胞/孔接种于96孔板中，同时加入不同浓度的化合物(40μM为最高浓度，按照1/4进行稀释，一共7个浓度)，共同孵育3天后，每孔加入10μl的CCK8试剂，孵育2小时后，通过酶标仪检测OD450，然后计算化合物对细胞活力的影响，计算IC 50值。2.2评价结果
表7 代表化合物对OCI-AML3细胞的抑制活性
化合物 OCI-AML3 IC 50(nM) 化合物 OCI-AML3 IC 50(nM)
A4-1a 81 B9-1e 274
A4-1d 169 B11-1b 47
A4-1i 91 B11-1c 289
B9-1d 602 Arctigenin 589
由评价结果可见，本发明化合物具有良好的抑制OCI-AML3细胞活性的作用，能用于制备治疗急性髓性白血病的药物。
3、化合物对人神经母细胞瘤细胞(NB-1)活力影响评价
3.1实验方法
NB-1细胞为悬浮细胞，培养于RPIM1640+10％FBS+1％PS培养液中，37℃，5％CO 2。NB-1按照20000个细胞/孔接种于96孔板中，同时加入不同浓度的化合物(40μM为最高浓度，按照1/4进行稀释，一共7个浓度)，共同孵育3天后，每孔加入10μL的CCK8试剂，孵育2小时后，通过酶标仪检测OD450，然后计算化合物对细胞活力的影响，计算IC 50值。
3.2评价结果
表8 代表化合物对NB-1细胞的抑制活性
化合物 NB-1 IC 50(nM) 化合物 NB-1 IC 50(nM)
A4-1a 424 A4-1d 546
A4-1i 433 B11-1b 626
B9-1e 175 B11-1c 446
Arctigenin 2380    
由评价结果可见，本发明化合物具有良好的抑制NB-1细胞活性的作用，能用于制备治疗神经胶质细胞瘤的药物。
4、化合物对衰老的人纤维母细胞(WI-38)和小鼠骨样细胞(MLO-Y4)的活力影响评价
4.1实验方法
4.1.1化合物对WI-38正常细胞和衰老细胞选择性实验：
WI-38细胞培养于DMEM(高糖，4.5g/L)培养液中(加入10％FBS，1％PS)。
化合物对WI-38衰老细胞活力影响的实验检测步骤如下：WI-38细胞按照20000个/孔接种于96孔板中，隔夜后每孔均加入200nM的阿霉素孵育3天，更换成正常培养液继续孵育1天，再更换成200nM的阿霉素孵育3天，即可成功诱导WI-38细胞衰老；随后加入不同浓度的化合物(40μM为最高浓度，按照1/4进行稀释，一共7个浓度)，共同孵育3天后，每孔加入MTT(5mg/ml)10μl，继续孵育4小时后，吸取培养液后每孔加入DMSO(150μl)，振荡孵育15分钟后，采用酶标仪检测OD490，然后计算化合物对细胞活力的影响，计算IC 50值。
4.1.2化合物对MLO-Y4正常细胞和衰老细胞选择性实验：
MOL-Y4细胞培养于MEM培养液中(加入10％FBS，1％PS)。
化合物对MOL-Y4衰老细胞活力影响的实验检测步骤如下：MOL-Y4细胞按照20000个/孔接种于96孔板中，隔夜后每孔均加入100nM的阿霉素孵育3天，更换成正常培养液继续孵育1天，再更换成100nM的阿霉素孵育3天，即可成功诱导MOL-Y4细胞衰老；随后加入不同浓度的化合物(40μM为最高浓度，按照1/4进行稀释，一共7个浓度)，共同孵育3天后，每孔加入MTT(5mg/ml)10μl，继续孵育4小时后，吸取培养液后每孔加入DMSO(150μl)，振荡孵育15分钟后，采用酶标仪检测OD490，然后计算化合物对细胞活力的影响，计算IC 50值。
4.2评价结果
表9 代表化合物对衰老细胞活力的抑制作用
化合物 WI-38 IC 50(nM) MOL-Y4 IC 50(nM)
A4-1a 38 40
A4-1e 1000 150
ABT263 5600 235
注：BCL-2抑制剂ABT263为阳性对照。
由评价结果可见，本发明化合物可有效地抑制衰老细胞的活力。由于衰老细胞不具有复制能力，因此细胞活力的降低即代表被药物诱导凋亡，也就是被清除。本发明化合物清除衰老细胞的作用优于现有的衰老细胞清除剂ABT263，能用于制备治疗衰老相关疾病的药物。
5、化合物对骨关节炎模型小鼠的药效评价
5.1实验原理和方法
原理：
小鼠膝关节的内侧半月板韧带切除术导致小鼠的膝关节半月板不稳定，从而诱导小鼠骨性关节炎。骨性关节炎的一个重要临床特征为关节疼痛、关节肿胀和活动受限，所以增加骨性关节炎小鼠的疼痛敏感性被认为对骨性关节炎具有治疗作用。因此，本实验对C57小鼠进行内侧半月板韧带切除术，诱导小鼠骨性关节炎，然后手术侧膝关节皮下注射药物持续八周，然后通过热板实验检测骨性关节炎小鼠的疼痛敏感性，小鼠膝关节的番红固绿染色实验，进而评价化合物对骨性关节炎的治疗作用。
方法：
1)采用内侧半月板韧带切除术导致内侧半月板不稳定(destabilization of the medical meniscus，DMM)的骨性关节炎模型小鼠。
A,8周龄的C57雄性小鼠麻醉后，75％乙醇将小鼠右侧后肢膝关节处消毒，然后用剪刀再右侧膝关节处将皮肤剪开2cm的切口。随后采用钝性分离右侧膝关节内侧处肌肉和滑膜组织，然后将髌骨采用钝器向外侧移位，暴露关节腔，在手术显微镜下钝性分离关节腔内脂肪垫，暴露膝关节内侧半月板韧带，然后通过手术刀片将内侧半月板韧带切断，能够明显感觉内侧半月板游离(可以滑动)。随后将髌骨复位，依次缝合肌肉和皮肤。手术后腹腔注射给予青霉素(100mg/kg/day)3天，预防细菌感染。
B，手术后当天小鼠苏醒后给药组给与化合物A4-1a(2mM,50μL/只)，其他组给与溶剂(生理盐水)。其后隔天给药一次，持续8周。
2)最后一次注射后，第二天进行热板实验，检测小鼠的疼痛敏感性。实验方法如下，小鼠于实验环境适应30分钟，将小鼠置于52℃的热板上，记录小鼠初次出现如舔足底、跳动等行为的时间，每只小鼠进行3次实验，然后进行统计学分析。
3)热板实验结束后，采用二氧化碳窒息致死，随后取小鼠右侧后肢膝关节(进行手术的关节)固定于多聚甲醛中，随后进行脱钙，然后进行石蜡切片，并对小鼠膝关节组织进行番红固绿染色或p16免疫染色，在显微镜下观察膝关节软骨组织形态，进行骨性关节炎评分(OA Score)和p16阳性面积测定，并进行统计学分析。
5.2评价结果
图1示出小鼠在热板实验中的反应时间；图2示出小鼠关节病理形态图片及OA评分；图3示出关节软骨免疫组化p16阳性率。
由评价结果可见，本发明化合物A4-1a能显著减少热板实验中骨关节炎小鼠的反应时间，表明有助于小鼠运动能力的恢复。在病理评价中，A4-1a能恢复受损关节软骨表面的光滑度和完整性，降低小鼠关节组织病理评分，降低软骨中衰老标记物p16的表达，表明化合物A4-1a可有效清除损伤诱导的衰老细胞、改善骨关节病理形态。
6、化合物对博来霉素诱导的特发性肺纤维化(IPF)小鼠的药效评价
6.1实验方法
实验动物：雄性C57小鼠，24-28g/只，70只。
博来霉素诱导肺纤维化模型及给药：65只C57小鼠，博来霉素按照2U/kg，麻醉后经口咽部吸入给药进行IPF造模，于给药前和给药后第5天测定每只小鼠对应的体重，选取体重有明显下降的小鼠作为造模成功的标准，选取20只成功造模的小鼠分为2组(分别：溶剂组——给予溶剂，A4-1a-30组——口服给药给予A4-1a 30mg/kg/day，A4-1a-10组——口服给药给予A4-1a 10mg/kg/day)，另外选取5只C57小鼠作为正常小鼠对照。于第5天开始给药，给药14天。每天记录小鼠的体重和生存率。
小鼠脏器处理：给药后第15天，小鼠麻醉后称重，处死小鼠，去小鼠肺组织，称重，小鼠肺指数的计算方法：小鼠肺组织重量/小鼠体重*1000；取肺组织中间1/3部分组织取出后固定于4％多聚甲醛中，用于后续石蜡切片及Masson染色。
小鼠肺组织Masson染色：按照常规方法将小鼠肺组织进行石蜡包埋后，切片进行masson染色。具体程序如下：切片脱蜡后，苏木素染色2分钟，蒸馏水2-3秒，分化液2-3秒，蒸馏水2-3秒，返蓝液2-3秒，蒸馏水2-4分钟，品红染色6分钟，蒸馏水2-3秒，磷钼酸2-3秒，苯胺蓝2分钟，1％乙酸2-3秒，100％乙醇2分钟，重复100％乙醇2分钟，二甲苯2分钟，重复二甲苯2分钟，中性树胶封片。
6.2评价结果
图4示出IPF小鼠给药后体重变化情况；图5示出小鼠给药后生存率变化情况；图6示出小鼠肺组织切片Masson染色及病理评分；图7示出小鼠肺组织衰老标记物p16基因水平；图8示出小鼠肺组织SASP中IL-6基因水平。
由评价结果可见，本发明化合物A4-1a能显著减缓IPF小鼠的体重降低，提高小鼠生存率，降低肺组织切片纤维化程度(Masson染色评分)、降低肺组织内衰老标记物p16水平和衰老相关的分泌表型IL-6，表明化合物对改善或治疗衰老相关疾病IPF具有十分显著的效果。7、化合物A4-1a对阿尔兹海默症(AD)小鼠的药效评价
7.1实验原理与方法
本发明中采用APP/PS1转基因的阿尔茨海默症(AD)模型小鼠。这类转基因小鼠高表达嵌和鼠/人的瑞典突变APP(Mo/HuAPP695swe)和人源的删除了第9个外显子的早老素1蛋白(presenilin，PS1-dE9)，这类转基因小鼠会在6个月的时候出现较明显的Aβ沉积并 且会在7个月的时候出现空间记忆力的损害。因此，本发明采用六个月大小的APP/PS1转基因的AD小鼠，灌胃给药(A4-1a,30mg/kg/day)三个月后通过Morris水迷宫实验检测A4-1a对转基因小鼠的记忆力损伤情况，以此类型老鼠的阴性小鼠为对照组，评价A4-1a对阿尔茨海默病的治疗作用。
1)APP/PS1双转基因AD模型小鼠进行繁殖。对子代小鼠的转基因型鉴定，通过采用对小鼠剪尾，然后PCR鉴定小鼠的APP/PS1的基因序列。以非转基因小鼠作为实验中的阴性对照小鼠。小鼠在标准条件下饲养(12/12小时明暗循环，足够水和食物，22℃的恒温，60％的湿度)。
2)AD模型小鼠给药：在小鼠6月大小时，20只转基因小鼠被随机分为2组(转基因溶剂组，转基因A4-1a-30mg/kg/day剂量组)，10只非转基因小鼠作为阴性对照组。A4-1a溶解于百分之二的吐温80中，灌胃给药90天，然后开始行为学实验检测(Morris水迷宫实验)。
3)小鼠每天进行三次训练，持续8天的时间。在训练小鼠面向池壁置于水中，60秒钟时间去寻找平台位置，小鼠在平台上停留15秒钟时间以帮助它记忆平台位置。在此期间，记录小鼠找到平台的时间即潜伏期。在第9天，在三次训练之后，小鼠进行平台寻找实验，潜于水面下的平台被撤除，然后让小鼠在水池中持续90秒钟去寻找平台，记录小鼠穿越平台次数作为其记忆力的评价指标，次数越多表明其记忆力越好。所有的动物实验操作都严格遵守《实验动物管理条例》。
7.2评价结果
如图9和图10所示，转基因小鼠(T-V)的潜伏期明显长于非转基因小鼠(NT-V)，而穿越平台次数明显少于非转基因小鼠，表明转基因小鼠的记忆力出现明显损伤，表明其AD模型正确。口服给予A4-1a 30mg/kg/day的转基因小鼠(T-A4-1a-30)潜伏期明显短于溶剂处理的转基因小鼠，而穿越平台次数明显多于溶剂处理的转基因小鼠，表明A4-1a能够逆转转基因小鼠出现的记忆力损伤表现。这些结果表明A4-1a能够起到很好治疗阿尔茨海默病效果。
8、化合物A5对四氯化碳诱导的肝纤维化小鼠的药效评价
8.1实验方法
实验动物：雄性C57小鼠，8周龄，40只。
建模及给药：400uL的四氯化碳(CCl 4)加入3.6mL的橄榄油中混匀然后按照0.1mL/20g进行腹腔注射，每周2次，持续六周。六周后30只小鼠分为3组，每组10只，分别采用灌胃方式给予生理盐水，A5剂量1mg/kg和A5剂量3mg/kg。另外，10只未进行诱导的小 鼠给予生理盐水作为对照组。持续给药3周后小鼠处死，取肝脏进行称重后，进行固定及包埋，剩余肝脏组织冻存，进行羟脯氨酸含量检测。
小鼠肝组织Masson染色：按照常规方法将小鼠肝组织进行石蜡包埋后，切片进行masson染色。具体程序如下：切片脱蜡后，苏木素染色2分钟，蒸馏水2-3秒，分化液2-3秒，蒸馏水2-3秒，返蓝液2-3秒，蒸馏水2-4分钟，品红染色6分钟，蒸馏水2-3秒，磷钼酸2-3秒，苯胺蓝2分钟，1％乙酸2-3秒，100％乙醇2分钟，重复100％乙醇2分钟，二甲苯2分钟，重复二甲苯2分钟，中性树胶封片。
小鼠肝组织羟脯氨酸含量检测：采用羟脯氨酸检测试剂盒进行(南京建成生物工程研究所)，简单介绍如下：称取肝脏组织40mg，加入碱裂解液0.5mL，95℃水解20分钟，然后加入酸碱度指示剂5μL，混匀，再加入调pH甲液0.5ml，混匀后呈红色，在缓慢加入调pH乙液至红色消失呈黄绿色。随后加入去离子水5mL混匀，然后加入活性炭20mg混匀后，3500转/分钟离心10分钟，取上清用于羟脯氨酸测定。按照试剂盒说明分别加入反应试剂一后，反应10分钟，再加入反应试剂二反应5分钟，最后加入反应试剂3后于60℃反应15分钟，然后于3500转/分钟离心15分钟，上清采用酶标仪于波长550nm测定吸光值。然后计算每100mg的肝脏组织中的羟脯氨酸含量。
8.2评价结果
如图11所示，CCl 4诱导并给予溶剂的小鼠肝脏中所含羟脯氨酸(HYP)高于正常对照组小鼠，具有统计学意义。而化合物A5灌胃给与1mg/kg/day和3mg/kg/day均能够明显地降低CCl 4诱导引起的羟脯氨酸含量增高，具有统计学意义。
如图12所示，CCl 4诱导的小鼠肝脏组织Masson染色阳性率，即肝纤维化面积相对正常组小鼠明显增加，具有统计学意义。而化合物A5灌胃给与1mg/kg/day和3mg/kg/day均能够明显地降低小鼠的肝纤维化程度，具有统计学意义。
以上结果表明，化合物A5对于改善或治疗肝纤维化具有较好的效果。
9、化合物A5对恶唑酮诱导的炎症性肠病小鼠的药效评价
9.1实验方法
实验动物：雄性C57小鼠，8周龄，30只。
建模及给药：3％的恶唑酮(Oxazolone)溶解于溶剂(丙酮：橄榄油，1：4)中，小鼠麻醉后，将小鼠肩部毛发剔除，然后滴150uL质量分数为3％的Oxazolone。在处理后的第8天，1％的Oxazolone溶解于溶剂(乙醇：灭菌水，1：1)，小鼠麻醉后，进行直肠给予1％的Oxazolone 150uL进行造模。造模小鼠分为两组，每组10只，一组给予化合物A5灌 胃1mg/kg/day，一组给予生理盐水，另外一组不进行造模的10只小鼠作为对照组。从造模当天开始，每天记录小鼠体重，直肠给药后第三天收集小鼠的粪便，观察其粪便松软程度，是否有出血。然后小鼠处死，取远端3cm左右的小鼠结肠进行固定。
小鼠肠炎疾病活动指数评分：根据小鼠体重减少程度(0:正常；1:1–5％；2:6–10％；3:11–18％；4:>18％)，粪便松软程度(0:正常；2：软；4：腹泻)，粪便出血程度(0:正常；2：隐血；4：血便)，然后各分数相加后除以三。
小鼠结肠组织伊红-苏木精染色：将小鼠结肠组织固定后，进行石蜡切片，然后进行伊红-苏木精染色。小鼠结肠炎症指数计算方法：根据小鼠结肠粘膜完整性(0:正常；1:粘膜损伤；2:粘膜下损伤；3:肌肉损伤)，炎症细胞浸润程度(0:正常；1:大量炎症细胞粘膜层浸润；2:炎症细胞浸润到粘膜下层；3:炎症细胞浸润透入肌层)，结肠的水肿程度(0:正常；1:轻度水肿；2中度水肿；3中度水肿)。
9.2评价结果
如图13所示，Oxazolone诱导的肠炎模型小鼠的疾病活动指数明显高于正常组，而灌胃给予化合物A5(1mg/kg/day)能够显著地降低肠炎疾病活动指数。
如图14所示，Oxazolone诱导的肠炎模型小鼠的结肠病理评分明显高于正常对照组小鼠，而灌胃给予A5(1mg/kg/day)能够明显地减少结肠病理评分。
以上结果表明化合物A5对治疗或缓解炎症性肠病变具有明显效果。
实施例8药代动力学评价
1、化合物A4-1a和牛蒡子苷元(Arctigenin)的口服药代动力学评价
1.1实验方法
30mg/kg的Arctigenin和化合物A4-1a分别口服给药给予禁食过夜的ICR小鼠(雄性，n＝3/时间点)。于给药后2小时喂食小鼠。分别于给药前及给药后1h、2h、4h、8h采集样，用肝素钠和磷酸二(4-硝基苯基)酯(BNPP)预处理的离心管中收集血液。离心后在4℃下以11,000rpm的转速离心5分钟，获得血浆并将其储存在-80℃下。接下来，将100mL甲醇/乙腈(1：1，v/v)添加到10mL血浆样品中，将其沉淀，涡旋振荡1分钟，离心(11,000rpm)5分钟。取20mL上清液和20mL甲醇/乙腈(1：1，v/v)的混合物通过LC-MS/MS分析化合物的浓度。肝脏组织并保存在-80℃直至分析。除去内含物后，结肠用含有BNPP的冷盐水快速洗涤，并保存在-80℃直至分析。分析方法：组织样品中加入10倍重量的MeOH/ACN(1:1，v/v)后用匀浆机在50Hz条件下匀浆120s后获得匀浆。将匀浆离心(11000rpm)5分钟并收集上清液。然后将20μL上清液重新溶解于20μL ACN/H2O(1:1，v/v)中后在LC-MS/MS中分析化合物的浓度。
1.2评价结果
图15示出化合物A4-1a和Arctigenin对小鼠口服30mg/kg后血浆、肝、结肠组织内药物浓度/时间变化曲线。
由评价结果可见，相比于Arctigenin，结构改造所得化合物A4-1a口服相同剂量后血浆和组织药物浓度显著提高，表明口服药代动力学性质获得了意想不到的改善。
2、化合物A4-1a的磷酸酯前药A5的口服药代动力学评价
2.1实验方法
口服给药化合物A5(30mg/kg)给予禁食过夜的ICR小鼠(雄性，n＝3/时间点)。于给药后2小时喂食小鼠。分别于给药前及给药后1h、2h、4h、8h采集样，用肝素钠和磷酸二(4-硝基苯基)酯(BNPP)预处理的离心管中收集血液。离心后在4℃下以11,000rpm的转速离心5分钟，获得血浆并将其储存在-80℃下。接下来，将100mL甲醇/乙腈(1：1，v/v)添加到10mL血浆样品中，将其沉淀，涡旋振荡1分钟，离心(11,000rpm)5分钟。取20mL上清液和20mL甲醇/乙腈(1：1，v/v)的混合物通过LC-MS/MS分析化合物A5以及A4-1a的浓度。心脏(血排空)、肝脏、肺组织并保存在-80℃直至分析除去内含物后，十二指肠和结肠用含有BNPP的冷盐水快速洗涤，并保存在-80℃直至分析。分析方法：组织样品中加入10倍重量的MeOH/ACN(1:1，v/v)后用匀浆机在50Hz条件下匀浆120s后获得匀浆。将匀浆离心(11000rpm)5分钟并收集上清液。然后将20μL上清液重新溶解于20μL ACN/H2O(1:1，v/v)中后在LC-MS/MS中分析化合物A5以及A4-1a的浓度。
2.2评价结果
图16示出化合物A5对小鼠口服30mg/kg后血浆及组织内前药A5(A)和原形A4-1a(B)的药物浓度/时间变化曲线。
由评价结果可见，磷酸酯前药A5口服后在体内能高效地代谢出原型化合物A4-1a，在血浆及各组织内均有较好的药物浓度，因此在制备预防或治疗本发明所述疾病的药物中同样具有潜在的应用价值。
总之，本发明基于牛蒡苷元(Arctigenin)进行结构改造得到通式I化合物并产生了意想不到的效果，或者提高了Complex I活性，或者提高了抑制肿瘤细胞活性，或者提高了清除衰老细胞活性，或者改善了口服药代动力学性质，或者在疾病动物模型中表现出了十分显著的药效，因此在预防和/或治疗与线粒体呼吸链复合物I活性或表达升高、或者线粒体氧化磷酸化作用增强相关的疾病中具有很好的应用潜力。

Claims (10)

  1. 一种式I所示的化合物、其对映异构体、非对应异构体、外消旋混合物、氘代化物及药学上可接受的盐，
    Figure PCTCN2022083003-appb-100001
    其中，
    Rx选自氢、羟基、卤素；
    R 0选自氢、卤素、C 1-C 6烷基、羟基取代的C 1-C 6烷基、C 1-C 6卤代烷基；
    R 1选自卤素、C 1-C 6卤代烷基、-N(R 5) 2、C 1-C 6烷氧基、C 1-C 6氘代烷氧基或-O-L-G；其中，L为C 1-6亚烷基，所述亚烷基的氢原子任选地以化学键允许的方式和数量被卤素、羟基、＝O所取代；G为卤素、C 1-C 6卤代烷基、-CN、-N(R 9) 2、-COOR 10、-P +(R 11) 3Y -、-N +(R 11) 3Y -、C 6-10芳基、C 6-10芳氧基、4-8元杂环基或5-10元杂芳基；所述芳基、芳氧基、杂环基和杂芳基未被取代或被1、2、3或4个选自下组的基团所取代：卤素、羟基、-N(R 12) 2、硝基、氰基、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、4-8元杂环基；
    R 2和R 3各自独立地为C 1-C 6烷基、C 1-C 6卤代烷基，或R 2和R 3连接起来并连同相连接的C形成未取代或取代的5-8元杂环基，所述取代是指被选自下组的一个或多个取代基取代：卤素、C 1-C 6烷基；
    R 4为氢或C 1-C 6烷基；
    各R 5各自独立地为氢、C 1-C 6烷基、-SO 2R 13、-COR 13；
    Ry为羟基或
    Figure PCTCN2022083003-appb-100002
    各R 9各自独立地为氢、C 1-C 6烷基、C 6-10芳基、5-10元杂芳基，所述芳基、杂芳基可任选地被1-4个选自下组的基团所取代：卤素、羟基、-N(R 12) 2、硝基、氰基、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、4-8元杂环基；
    各R 11各自独立地选自C 1-C 6烷基和C 6-10芳基，所述芳基任选地被1-4个选自下组的基团所取代：卤素、C 1-6烷基、C 1-6烷氧基；
    R 8、R 10、R 12各自独立地选自氢、C 1-C 6烷基；
    R 13为C 1-C 6烷基；
    Y -为卤离子、甲酸根离子、乙酸根离子、三氟乙酸根离子或氢氧根离子；
    m为0、1、2或3；各Q独立地为卤素或C 1-C 6烷基；
    条件是当R 0、R 4、R x均为氢且Ry为羟基时，R 1不为C 1-C 6烷氧基。
  2. 如权利要求1所述化合物，其特征在于，所述化合物具有如下式(IIa)：
    Figure PCTCN2022083003-appb-100003
    其中，各取代基定义与权利要求1中相同。
  3. 如权利要求1所述化合物，其特征在于，所述化合物具有如下式(IIb)：
    Figure PCTCN2022083003-appb-100004
    其中，各取代基定义与权利要求1中相同。
  4. 如权利要求1所述化合物，其特征在于，R 1选自卤素、C 1-C 4卤代烷基、-N(R 5) 2、C 1-C 4烷氧基、C 1-C 4氘代烷氧基或-O-L-G；其中，L为C 1-4亚烷基，所述亚烷基上的1、2或3个氢原子以化学键允许的方式被卤素、羟基、＝O所取代；G为卤素、C 1-C 4卤代烷基、-CN、-N(R 9) 2、-COOR 10、-P +(R 11) 3Y -、-N +(R 11) 3Y -、苯基、苯基氧基、4-6元杂环基或6-8元杂芳基；所述苯基、苯基氧基、杂环基和杂芳基未被取代或被1、2或3个选自下组的基团所取代：卤素、羟基、-N(R 12) 2、硝基、氰基、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、4-8元杂环基；
    其中，各R 5各自独立地为氢或C 1-C 4烷基；各R 9各自独立地为氢或C 1-C 4烷基；R 10为氢或C 1-C 4烷基；各R 11各自独立地为C 1-C 4烷基或苯基；各R 12各自独立地为氢或C 1-C 4烷基。
  5. 如权利要求1所述化合物，其特征在于，Ry为羟基或-OP(O)(OH) 2；
    R 0为氢或C 1-C 6烷基；
    R 2和R 3各自独立地为C 1-C 4烷基；
    R 1为C 1-C 4烷氧基或-O-L-G；其中，L为C 1-4亚烷基；G为苯基或C 1-C 4卤代烷基。
  6. 如权利要求1所述化合物，其特征在于，所述化合物选自：
    Figure PCTCN2022083003-appb-100005
    Figure PCTCN2022083003-appb-100006
    Figure PCTCN2022083003-appb-100007
    Figure PCTCN2022083003-appb-100008
  7. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1所述的化合物、其对映异构体、非对应异构体、外消旋混合物、氘代化物及药学上可接受的盐；和
    药学上可接受的载体。
  8. 如权利要求1所述的化合物、其对映异构体、非对应异构体、外消旋混合物、氘代化物及药学上可接受的盐或者权利要求7所述的药物组合物的用途，其特征在于，
    用于制备线粒体呼吸链复合体I抑制剂；
    用于制备预防和/或治疗与线粒体呼吸链复合物I活性或表达升高相关的疾病的药物；
    用于制备预防和/或治疗与线粒体氧化磷酸化作用增强相关的疾病的药物；
    用于制备预防和/或治疗与细胞衰老相关的疾病的药物；
    用于制备衰老细胞清除剂；或
    用于制备预防和/或治疗肿瘤的药物。
  9. 如权利要求8所述的用途，其特征在于，所述肿瘤选自：急性髓性白血病、神经胶质细胞瘤、淋巴瘤、胰腺癌、子宫癌、乳腺癌、非小细胞肺癌、肝细胞癌。
  10. 如权利要求8所述的用途，其特征在于，所述与细胞衰老相关的疾病选自：器官纤维化疾病、慢性肺病、慢性肾病、慢性肝病、骨关节炎、神经退行性疾病、炎症性肠病、动脉粥样硬化、青光眼、白内障、黄斑变性、糖尿病、糖尿病视网膜病变、色斑、少肌症。
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