WO2023116724A1 - 新型一叶萩碱二聚体及其制备方法和应用 - Google Patents

新型一叶萩碱二聚体及其制备方法和应用 Download PDF

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WO2023116724A1
WO2023116724A1 PCT/CN2022/140449 CN2022140449W WO2023116724A1 WO 2023116724 A1 WO2023116724 A1 WO 2023116724A1 CN 2022140449 W CN2022140449 W CN 2022140449W WO 2023116724 A1 WO2023116724 A1 WO 2023116724A1
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compound
dimer
monophyllin
pharmaceutically acceptable
formula
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French (fr)
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叶文才
师蕾
陈卫民
王英
胡利军
李刘任
马暄越
廖玉梅
庄晓吉
黄志星
黄小杰
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暨南大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

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  • the invention relates to the field of medicine, in particular to a medicine for treating Alzheimer's disease.
  • a medicine for treating Alzheimer's disease In particular, it relates to the application of the novel monophyllin dimer in the preparation of drugs for treating Alzheimer's disease.
  • AD Alzheimer's disease
  • a ⁇ ⁇ -amyloid
  • AD is the most common type of senile dementia, accounting for 60% to 80% of all dementia patients, and has become the third leading cause of death in the elderly after cardiovascular disease and cancer.
  • Epidemiological surveys show that the global incidence of AD continues to rise. It is estimated that by 2050, the number of AD patients will exceed 150 million. This is undoubtedly a public health and health problem that cannot be ignored by all centuries. However, since the pathophysiological changes of AD involve many factors, there is still no specific treatment drug so far.
  • the FDA acetylcholinesterase inhibitors
  • AChEIs acetylcholinesterase inhibitors
  • these drugs can restore the level of acetylcholine in the brain of AD patients to a certain extent, temporarily delay the course of the disease, and improve cognitive function.
  • the second category is N-methyl-D-aspartic acid (N-methyl-D-aspartic acid, NMDA) receptor antagonists, the representative drug is memantine.
  • This type of drug can reduce the neuronal damage caused by excessive excitation of the glutamatergic system, improve learning and memory functions to a certain extent, and relieve symptoms of AD patients in the middle and late stages (Cummings JL, et al, Alzheimers Res Ther, 2014, 6(4):37).
  • the treatment strategies of these drugs mainly focus on the compensation of neurotransmitters and the inhibition of specific enzymes or receptors, all of which are mainly aimed at slowing down the symptoms of AD, and cannot reverse the severe synapse loss and Neuron damage phenomenon, so it is impossible to fundamentally reverse or stop the development of the disease.
  • AD Alzheimer's disease
  • Protein biosynthesis determines the accurate expression of proteins in various physiological activities of cells, and is also a key regulatory site for cells to adapt and survive under stress. Since a neuron is a specialized cell with a high demand for energy metabolism, its protein synthesis needs to dynamically occur on dendrites or synapses far away from the cell body, thereby determining the accurate occurrence of synapse formation and plasticity, maintaining The learning and memory functions of the brain.
  • AD animal models it has been confirmed that by restoring or partially restoring the protein synthesis level in neurons, neurodegeneration in model animals can be effectively alleviated or reversed (Hernandez-Ortega K, et al, Brain Pathol, 2016, 26(5 ):593–605; Yang WZ, et al, Neurobiol Aging, 2016, 41:19–24). Therefore, by restoring the protein synthesis level of neurons, it is expected to increase the expression of key synaptic proteins in the brain of AD patients and improve the function of synapses, thus becoming an effective new strategy for the treatment of AD.
  • Chinese patent CN104761572B discloses that a hagi-type alkaloid dimer compound or a pharmaceutically acceptable salt thereof can be used in the preparation and treatment of neurodevelopmental abnormalities, nerve damage, neurodegenerative diseases and learning and memory disorders.
  • trichopine dimer compounds such as SN3-L6
  • SN3-L6 Some simple types of trichopine dimer compounds can significantly up-regulate the synthesis level of neuronal proteins, and at the same time have the effect of promoting neuronal differentiation and enhancing synaptic function (Tang G, et al, ACS Chem Neurosci, 2016, 7:1442–1451; Liao Y, et al, Front Pharmacol, 2018, 9:290), but these compounds are rapidly eliminated in animals, which affects their good therapeutic effect in vivo.
  • the purpose of the present invention is to overcome the deficiencies of the prior art and provide a novel monophyllin dimer compound that can be used for the treatment of Alzheimer's disease.
  • a novel monophyllin dimer which has the structural formula shown in general formula I or its pharmaceutically acceptable salt or its stereoisomer or its prodrug molecule:
  • the linking group Linker is selected from:
  • R1 is a hydrogen atom
  • X selected from or n is 1, 2, 3 or 4.
  • the compound represented by the general formula II includes, but is not limited to:
  • the compound represented by the general formula III includes, but is not limited to:
  • the present invention relates to a pharmaceutical composition, which contains a therapeutically effective dose of abapine dimer represented by general formulas I, II and III, or a pharmaceutically acceptable salt thereof or a stereoisomer or its precursor Drug molecules, and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • a pharmaceutical composition which contains a therapeutically effective dose of abapine dimer represented by general formulas I, II and III, or a pharmaceutically acceptable salt thereof or a stereoisomer or its precursor Drug molecules, and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the present invention relates to the monophyllin dimer represented by the general formulas I, II and III, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, or its pharmaceutical combination containing therapeutically effective dosage
  • Prodrug means a prodrug converted into the structure of the compound involved in the present application and the pharmaceutically acceptable salt thereof in vivo.
  • “Pharmaceutical composition” means a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, and other components such as a physiologically/pharmaceutically acceptable carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
  • “Pharmaceutically acceptable salt” refers to the salt of the compound of the present invention, which is safe and effective when used in mammals, and has proper biological activity.
  • the preparation method of the compound shown in general formula II of the present invention comprises the following steps:
  • azide N10a or N10b (1.0 equiv.) was dissolved in stirred DMSO solvent, and copper (2 equiv.), copper sulfate pentahydrate (0.05 equiv.) and diacetylenic compound (0.27 equiv.) were added successively.
  • copper (2 equiv.), copper sulfate pentahydrate (0.05 equiv.) and diacetylenic compound (0.27 equiv.) were added successively.
  • ethyl acetate was added, and the filtrate was obtained by filtration.
  • the filtrate was washed 3 times with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product.
  • the obtained crude product was separated and purified by silica gel column chromatography to obtain compound II of general formula.
  • X is as defined in claim 1.
  • the preparation method of the compound shown in general formula III of the present invention comprises the following steps:
  • the present invention develops the novel monophyllin dimer compound with triazole as the linker by changing the linker (Linker), and these novel dimer compounds show a simpler type of monophyllin Dimeric compounds (such as SN3-L6) have better activity in inducing neural differentiation and promoting protein synthesis, and have better stability in cells and animals, and the cognitive function of AD model mice is significantly improved after administration , is expected to develop into an effective drug for the treatment of AD.
  • Linker linker
  • Figure 1 The role of the novel monophyllin dimer compound (25 ⁇ M) in promoting the synthesis of new proteins in Neuro-2a cells
  • NMR nuclear magnetic resonance
  • MS mass spectroscopy
  • MS was determined with a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).
  • the known starting materials of the present invention can be adopted or synthesized according to methods known in the art, or can be purchased from Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc), Bailingwei, Anaiji, Da Swiss Chemicals and other companies.
  • Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a volume of about 1 L.
  • the solution refers to an aqueous solution.
  • reaction temperature is room temperature, which is 20°C to 30°C.
  • the monitoring of the reaction process in the embodiment adopts thin-layer chromatography (TLC), and the developing agent used in reaction, the eluent system of the column chromatography that separation and purification compound adopts and the developing agent system of thin-layer chromatography include: A: Dichloromethane/methanol system; B: n-hexane/ethyl acetate system; C: petroleum ether/ethyl acetate system; D: acetone; E: dichloromethane/acetone system; F: ethyl acetate/dichloromethane system ; G: ethyl acetate/dichloromethane/n-hexane; H: ethyl acetate/dichloromethane/acetone.
  • the volume ratio of the solvent is adjusted according to the polarity of the compound, and it can also be adjusted by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.
  • the azide compound N10a was dissolved in a stirred DMSO (2 mL) solvent, and copper (127 mg, 2 mmol), copper sulfate pentahydrate (13 mg, 0.05 mmol) and 1,5-hexadiyne (26 ⁇ L, 0.27 mmol) were successively added ).
  • ethyl acetate (10 mL) was added, and the filtrate was obtained by filtration.
  • the filtrate was washed 3 times with water (3 ⁇ 10 mL), washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product.
  • the obtained crude product was separated and purified by silica gel column chromatography to obtain compound 1 as a white solid (total yield 51%).
  • the azide compound N10a was used as the starting material to react with 1,6-heptadiyne to obtain white solid compound 3 (69% yield).
  • the azide compound N10b was used as the starting material to react with 1,6-heptadiyne to obtain white solid compound 4 (54% yield).
  • the azide compound N10a was used as the starting material to react with 1,7-octadiyne to obtain white solid compound 5 (75% yield).
  • the azide compound N10b was used as the starting material to react with 1,7-octadiyne to obtain white solid compound 6 (60% yield).
  • azide compound N10a was used as the starting material to react with propargyl ether to obtain white solid compound 7 (57% yield).
  • azide compound N10b was used as the starting material to react with propargyl ether to obtain white solid compound 8 (57% yield).
  • azide compound N10a was used as the starting material to react with compound N4 to obtain white solid compound 9 (56% yield).
  • azide compound N10b was used as the starting material to react with compound N4 to obtain white solid compound 10 (64% yield).
  • Example 12 The role of novel monophyllin dimer compounds in promoting neural differentiation
  • Neuro-2a cells purchased from American type culture collection cell bank
  • the cells grow to 60-70% for subculture, absorb the culture medium in the culture dish, add an appropriate amount of PBS to wash, and then digest with 0.25% trypsin for 45 seconds. After the adherent cells are spherical, add growth medium to stop digestion, and mix well. Passage according to 1:10, every three days.
  • the cell planting density is 2 ⁇ 104 cells/35mm culture dish or 1 ⁇ 104 cells/well (12-well plate), and the growth medium is cultured for 24 hours and then replaced with a differentiation medium (MEM+0.5% FBS +100 U/mL of penicillin and 100 ⁇ g/mL of streptomycin), and added a concentration of 25 ⁇ M of abaline dimer compounds for 48 h.
  • a differentiation medium MEM+0.5% FBS +100 U/mL of penicillin and 100 ⁇ g/mL of streptomycin
  • the differentiation morphology and neurites of the nerve cell lines were observed by immunofluorescence staining, and the specific steps were as follows:
  • 3Discard the blocking solution add the primary antibody dilution containing ⁇ -tubulin III antibody (1:3000) (PBS containing 1% bovine serum albumin, 1% sheep serum albumin and 0.4% Triton X-100 ), incubate at room temperature for 1 h or overnight at 4°C;
  • Table 1 The activity of novel monophyllin dimer compounds in promoting the differentiation of Neuro-2a cells at a dose of 25 ⁇ M
  • the data of the experimental results are represented by mean ⁇ S.E.M (standard error of the mean), and there are three independent experiments, at least 100 cells are analyzed in each group, and the unpaired t-test method is used for statistical processing, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • the data of the experimental results are represented by mean ⁇ S.E.M (standard error of the mean), and there are three independent experiments, at least 100 cells are analyzed in each group, and the unpaired t-test method is used for statistical processing, *P ⁇ 0.05, **P ⁇ 0.01.
  • Example 13 The new-type monophyllin dimer compound promotes the synthesis of newborn protein in nerve cells
  • Experimental method use puromycin (puromycin) labeling method to measure the synthetic amount of newborn protein in nerve cells after treatment with the dimer compound of monophyllin.
  • Plant Neuro-2a cells in a 35mm culture dish at a density of 6 ⁇ 105 cells/35mm culture dish. After culturing overnight in a constant temperature incubator containing 5% CO2 at 37°C, add a mixture of adenine dimer compounds Homogenize the mixture so that the final concentration is 25 ⁇ M, add an equal volume of DMSO to the control group, and incubate for 1 hour at 37° C. in a constant temperature incubator containing 5% CO2. Add 1 ⁇ M puromycin to the culture medium, and continue culturing for 0.5 h at 37° C.
  • Said Figure 1 is a histogram of the content of puromycin-labeled nascent protein detected by Western blot.
  • Neuro-2a cells were treated with 25 ⁇ M compound for 1 h, and then treated with 1 ⁇ M puromycin for 0.5 h, and the DMSO group was set as the control group.
  • the data of the experimental results are expressed as mean ⁇ S.E.M (standard error of the mean), with three independent experiments and one-way ANOVA test method for statistical processing, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Said Figure 2 is a histogram of the content of puromycin-labeled nascent protein detected by Western blot.
  • Neuro-2a cells were treated with 1 ⁇ M or 10 ⁇ M compound for 1 h, and then treated with 1 ⁇ M puromycin for 0.5 h, and the DMSO group was set as the control group.
  • the data of the experimental results are represented by mean ⁇ S.E.M (standard error of the mean), with three independent experiments, and one-way ANOVA test method for statistical processing, *P ⁇ 0.05, different compounds vs DMSO.
  • compound 3 has the best activity and the strongest ability to enter cells, so we choose compound 3 as a representative compound to study its metabolism in animals.
  • mice were intraperitoneally injected with pentobarbital sodium, and after the anesthesia was complete, the eyeballs were removed to collect blood, which was collected in a 1.5mL centrifuge tube containing anticoagulant, and mixed by inverting gently. Take 500 ⁇ L of blood; centrifuge at 3800 rpm at 4°C for 20 min; take 300 ⁇ L of the upper layer of plasma, add 1 mL of extractant (ethyl acetate), and vortex for 5 min;
  • Example 16 Compound 3 improves the memory cognitive function of AD model mice in the new object recognition test
  • the new object recognition test is a learning and memory test method established based on the principle that rodents have an innate tendency to explore new objects, and measures short-term or long-term memory and cognitive functions of animals.
  • This example uses APP1/PS1 double transgenic mice, which are classic AD model mice. Cognitive impairment begins to appear at the age of 4 to 5 months and gradually worsens.
  • mice male APP1/PS1 mice were randomly divided into groups (10 mice in each group), administered with compound 3, SN3-L6 or vehicle, and administered intragastrically at the age of 7 months, once a day for 4 weeks, Compound 3 and SN3-L6 were provided with three dosages (6.25mg/kg, 12.5mg/kg and 25mg/kg), and the solvent was normal saline (adding 5% Tween-80 to aid dissolution), and each mouse was 10 ⁇ L/g gavage corresponding volume of drugs or solvents.
  • WT male wild mice of the same age was set up and given a solvent. After the drug administration, the new object recognition test is carried out.
  • the experiment is mainly divided into the adaptation phase, the training phase and the testing phase:
  • mice Put the mice into the experimental device (40 ⁇ 40 ⁇ 40cm white plastic box) without any objects one by one, let them explore freely for 5 minutes to adapt to the experimental environment, and reduce the interference of animals exploring the new environment on the experiment;
  • Training phase Glue two identical objects (white plastic bottle caps, 4 cm in diameter and 2.8 cm in height) on the bottom plate of the experimental device with double-sided tape, and put the mouse into the experimental device with its back facing the two objects , make it explore freely for 10 minutes and record it with a video system, and after the end, take the animal out and put it back into the cage. After an interval of 1h, the test period experiment was carried out;
  • Test phase Replace one of the objects with an object of a different shape (square building blocks, 4.6 ⁇ 2.2 ⁇ 3.5cm).
  • the original object is called an old object or a familiar object
  • the new object is called a new object or a novelty object.
  • mice The total moving distance of the mice and the time spent sniffing different objects were analyzed with Topscan 3.0 software.
  • Said Fig. 3 is the novel object recognition ability shown by different groups of mice in the test phase.
  • new is the time of sniffing new objects
  • old is the time of sniffing old objects
  • recognition index time of sniffing new or old objects/total time of sniffing new and old objects.
  • Example 17 Compound 3 improves the spatial learning and memory function of AD model mice in the Morris water maze test
  • Training stage Inject water containing titanium dioxide (to reduce underwater visibility) into a circular water tank (diameter 120cm, depth 45cm), and keep the temperature at 25°C. Specify two mutually perpendicular major axes to divide the pool into four equal quadrants, and specify the intersection points of these axes with the edge of the pool as North (N), South (S), East (E), and West (W). Place a circular platform (diameter 10cm, height 30cm) in the center of a certain quadrant (target quadrant), and paste different patterns on the walls corresponding to different quadrants as maze clues for spatial learning. The training lasted for 7 days, 3 times a day (90s per group), with a 30min interval between each training session.
  • Test stage remove the platform on the 8th day (24 hours after the last test), put the mouse into the water facing the pool wall, and record its motion within 90s with a video system;
  • mice The residence time and swimming speed of mice in different quadrants were analyzed by Topscan 3.0 software.
  • Said Figure 4 shows the residence time of different groups of mice in the target quadrant and other quadrants respectively during the test phase.
  • Target is the residence time of mice in the target quadrant
  • Average of others is the residence time of mice in other quadrants.

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Abstract

本公开涉及医药领域，具体涉及新型一叶萩碱二聚体或其药学上可接受的盐在制备用于治疗阿尔茨海默病药物中的应用。所述新型一叶萩碱二聚体，具有通式（I）所示的结构式或其药学上可接受的盐或其立体异构体或其前药分子。式中：连接基团Linker选自式（ A）式（B）；X选自式（ II）或者式（ III）；n为1、2、3或4。

Description

新型一叶萩碱二聚体及其制备方法和应用 技术领域
本发明涉及医药领域，涉及治疗阿尔茨海默病的药物。具体涉及新型一叶萩碱二聚体在制备用于治疗阿尔茨海默病药物中的应用。
背景技术
阿尔茨海默病(Alzheimer’s disease,AD)，俗称老年痴呆症，是一种以进行性认知功能障碍和记忆损害为特征的中枢神经系统退行性疾病，常见的临床症状为进行性记忆障碍、认知障碍、人格障碍等，脑部病理特征主要为大量神经元丢失、β淀粉样蛋白(Aβ)组成的老年斑及Tau蛋白组成的神经缠结。AD是老年痴呆中最常见的类型，占所有痴呆症患者的60％～80％，现已成为继心血管疾病和癌症之后导致老年人死亡的第三大病因。流行病学调查显示，AD在全球的发病率持续上升，预计到2050年，患者数量将超过1.5亿，这无疑是全人类不容忽视的公共卫生与健康问题。然而，由于AD的病理生理学改变涉及多方面因素，迄今仍无特效治疗药物。
目前，FDA批准上市的抗AD药物主要有两大类，第一类是乙酰胆碱酯酶抑制剂(acetylcholinesterase inhibitors,AChEIs)，如多奈哌齐、加兰他敏、他克林、利凡斯的明等。该类药物通过抑制乙酰胆碱水解，可在一定程度上恢复AD病人脑内乙酰胆碱的水平，暂时延缓病程，改善认知功能。第二类是N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid,NMDA)受体拮抗剂，代表药物是美金刚。该类药物可减轻由于谷氨酸能系统过度兴奋而导致的神经元损伤,并可一定程度地改善学习记忆功能，缓解中晚期AD患者的症状(Cummings JL,et al,Alzheimers Res Ther,2014,6(4):37)。然而，这些药物的治疗策略主要集中在神经递质的代偿和特定酶或受体的抑制上，均以减缓AD的症状为主，并不能逆转病程中晚期所出现的严重神经突触丢失和神经元损伤现象，因此无法从根本上逆转或阻止病情的发展。大量研究表明，在AD病程中，神经突触丢失或功能异常等早期过程可逐渐演变为神经元的整体损伤和凋亡，从而导致行使认知等功能的神经环路被破坏，而恢复神经功能则需要神经突触的重塑以及相关神经环路的重构。近年来，针对拮抗Aβ蛋白和Tau蛋白等传统致病因素的AD药物研发相继宣告失败，最近，针对清除Aβ的单抗药物aducanumab虽获FDA批准，但其改善认知的效果仍存在颇多争议。其中，部分原因是由于在AD的中后期阶段，单纯清除Aβ蛋白或Tau蛋白等有害物质已不能修复被损害的神经突触及其所组成的神经环路(Ferreira ST,et al,Front Cell Neurosci,2015,9:191；Overk CR,et al,Biochem Pharmacol,2014,88(4):508–516)。因此，开发促进或保护神经突触功能的药物或干预方法，是AD药物研发的重要新途径。
蛋白质的生物合成决定了细胞在各个生理活动中蛋白质的准确表达，也是细胞在应激状态下适应和存活的一个关键调控位点。由于神经元是一种对能量代谢有高度需求的特化细胞，其蛋白质合成需要动态地发生在远离细胞体的树突或神经突触上，进而决定神经突触形成及可塑性的准确发生，维持着大脑的学习记忆等功能。当蛋白质合成降低时，重要的突触蛋白如谷氨酸受体、突触骨架蛋白等蛋白质合成受阻，影响神经突触的结构和功能，从而导致多种神经功能障碍(Tom DS,et al,Neuron,2014,81(4):958–958；Buffington SA,et al,Annu Rev Neurosci,2014,37:17–38)。近年来，蛋白质合成的异常或持续抑制已成为AD等神经系统疾病普遍被认可的病理机制之一。AD病人和模型动物的脑组织样本中蛋白质的合成水平明显降低，蛋白质稳态被破坏，导致重要的突触蛋白含量不足。在AD动物模型中，已证实通过恢复或部分恢复神经元中蛋白质的合成水平，可有效缓解或逆转模型动物的神经退行性病变(Hernandez-Ortega K,et al,Brain Pathol,2016,26(5):593–605；Yang WZ,et al,Neurobiol Aging,2016,41:19–24)。因此，通过恢复神经元的蛋白质合成水平，有望提高AD患者脑内关键突 触蛋白的表达，改善神经突触的功能，从而成为有效治疗AD的新策略。
但这些化合物在动物体内代谢消除较快，影响了其在体内发挥良好的治疗效果。中国专利CN104761572B公开了一种一叶萩型生物碱二聚体类化合物或其可药用盐可应用于制备治疗神经发育异常、神经损伤、神经退行性疾病和学习记忆障碍。一些简单类型的一叶萩碱二聚体类化合物(如SN3-L6)可显著上调神经元蛋白质的合成水平，同时具有促进神经元分化和增强神经突触功能的作用(Tang G,et al,ACS Chem Neurosci,2016,7:1442–1451；Liao Y,et al,Front Pharmacol,2018,9:290)，但这些化合物在动物体内代谢消除较快，影响了其在体内发挥良好的治疗效果。
因此，利用开发一种能解决上述技术问题的改善一叶萩碱二聚体类化合物治疗AD效果的药物是非常必要的。
发明内容
本发明的目的是克服现有技术的不足而提供一种可以用于治疗阿尔茨海默病的新型一叶萩碱二聚体类化合物。
本发明是通过以下技术方案予以实现的：
新型一叶萩碱二聚体，其具有通式I所示的结构式或其药学上可接受的盐或其立体异构体或其前药分子：
Figure PCTCN2022140449-appb-000001
式中：
连接基团Linker选自：
Figure PCTCN2022140449-appb-000002
R1为氢原子；
X选自
Figure PCTCN2022140449-appb-000003
或者
Figure PCTCN2022140449-appb-000004
n为1、2、3或4。
在本发明的一个优选的实施方案中，所述的通式II所示的化合物包括，但不限于：
Figure PCTCN2022140449-appb-000005
Figure PCTCN2022140449-appb-000006
在本发明的另一个优选的实施方案中，所述的通式I所示的化合物，其中Linker＝B时，其具有通式III所示的结构式或其药学上可接受的盐或其立体异构体或其前药分子：
Figure PCTCN2022140449-appb-000007
在本发明的一个优选的实施方案中，所述的通式III所示的化合物包括，但不限于：
Figure PCTCN2022140449-appb-000008
本发明涉及一种药物组合物，其含有治疗有效剂量的通式I、II和III所示的一叶萩碱二聚体，或其药学上可接受的盐或其立体异构体或其前药分子，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明涉及含有治疗有效剂量的通式I、II和III所示的一叶萩碱二聚体，或其药学上可接受的盐或其立体异构体或其前药分子，或其药物组合物在制备用于治疗阿尔茨海默病药物中的应用。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
“前药”表示在体内转变为本申请所涉及的化合物及其药学可接受的盐的结构的前药。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“可药用盐”是指本发明化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性。
本发明中化合物的合成方法
为了完成本发明的目的，本发明采用如下技术方案：
方案一：
本发明通式II所示的化合物的制备方法包括以下步骤：
Figure PCTCN2022140449-appb-000009
常温下，将一叶萩碱(1.0equiv.)溶于搅拌的二氯甲烷溶剂中，先后加入三甲基硅基叠氮(5.0equiv.)、醋酸(5.0equiv.)和DBU(0.05equiv.)。升温至35℃后，反应8小时，有机溶剂经减压浓缩得到粗产物。所得粗产物经硅胶柱色谱分离纯化，得到一对非对映异构体叠氮化合物N10a和N10b。随后，将叠氮化合物N10a或N10b(1.0equiv.)溶于搅拌的DMSO溶剂中，先后加入铜(2equiv.)、五水硫酸铜(0.05equiv.)和二炔类化合物(0.27equiv.)。避光反应5小时后，加入乙酸乙酯，过滤得到滤液。滤液用水洗3次，饱和食盐水洗，无水硫酸钠干燥，过滤和减压浓缩得到粗产物。所得粗产物经硅胶柱色谱分离纯化，得到通式化合物II。
X如权利要求1中所定义。
方案二：
本发明通式III所示的化合物的制备方法包括以下步骤：
Figure PCTCN2022140449-appb-000010
将叠氮化合物N10a或N10b(1.0equiv.)溶于搅拌的DMSO溶剂中，先后加入铜(2.0equiv.)、五水硫酸铜(0.05equiv.)和通式化合物III-1(0.27equiv.)。避光反应5小时后，加入乙酸乙酯，过滤得到滤液。滤液用水洗3次，饱和食盐水洗，无水硫酸钠干燥，过滤和减压浓缩得到粗产物。所得粗产物经硅胶柱色谱分离纯化，得到通式化合物III。
本发明的有益效果是：
本发明通过改变连接基团(Linker)，发展了以三氮唑作为连接基团的新型一叶萩碱二聚体类化合物，这些新型二聚体类化合物显示出较简单类型的一叶萩碱二聚体化合物(如SN3-L6)更优的诱导神经分化及促进蛋白质合成的活性，且在细胞及动物体内均具有更好的稳定性，给药后AD模型小鼠的认知功能明显改善，可望发展成为AD的有效治疗药物。
附图说明
图1.新型一叶萩碱二聚体类化合物(25μM)促进Neuro-2a细胞新生蛋白质合成的作用
图2.化合物3在低浓度下促进Neuro-2a细胞新生蛋白质合成的作用
图3.化合物3改善AD模型小鼠在新物体识别测试中的记忆认知功能
图4.化合物3改善AD模型小鼠在Morris水迷宫测试中的空间学习记忆功能
具体实施方式
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10-6(ppm)给出。NMR的测定是用Bruker AVANCE-300和Bruker AVANCE-400核磁共振波谱仪，测定溶剂为氘代氯仿(CDCl3)，内标为四甲基硅烷(TMS)。
MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商：Thermo，型号：Finnigan LCQ advantage MAX)。
柱层析一般使用烟台黄海硅胶200～300目硅胶为载体。
本发明的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自Acros Organics、Aldrich Chemical Company、韶远化学科技(Accela ChemBio Inc)、百灵威、安耐吉、达瑞化学品等公司。
实施例中无特殊说明，反应均在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃～30℃。
实施例中的反应进程的监测采用薄层色谱法(TLC)，反应所使用的展开剂，分离纯化化合物采用的柱层析的洗脱剂体系和薄层色谱法的展开剂体系包括：A：二氯甲烷/甲醇体系；B：正己烷/乙酸乙酯体系；C：石油醚/乙酸乙酯体系；D：丙酮；E：二氯甲烷/丙酮体系；F：乙酸乙酯/二氯甲烷体系；G：乙酸乙酯/二氯甲烷/正己烷；H：乙酸乙酯/二氯甲烷/丙酮。溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和醋酸等碱性或酸性试剂进行调节。
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，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
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。
以下实施例制备得到的具体化合物均包括但不限于出现在前面表格所述1～10中。
实施例1化合物N1～N6的通用制备方法
Figure PCTCN2022140449-appb-000011
常温下，将一叶萩碱(217mg,1mmol)溶于搅拌的二氯甲烷(5mL)和甲醇(0.5mL)的混合溶剂中，先后加入磷酸钾(K 3PO 4,64mg,0.3mmol)和相应的一级胺(R-NH 2,5mmol)。升温至35℃后，反应8小时。有机溶剂经减压浓缩得到粗产物。所得粗产物经硅胶柱色谱分离纯化，得到相应的化合物N1～N6。
实施例2化合物1的制备
Figure PCTCN2022140449-appb-000012
常温下，将一叶萩碱(2.17g,10mmol)溶于搅拌的二氯甲烷(50mL)溶剂中，先后加入三甲基硅基叠氮(6.58mL,50mmol)、醋酸(2.86mL,50mmol)和DBU(75L,0.5mol)。升温至35℃后，反应8小时，有机溶剂经减压浓缩得到粗产物。所得粗产物经硅胶柱色谱分离纯化得到一对非对映异构体叠氮化合物N10a和N10b。随后，将叠氮化合物N10a溶于搅拌的DMSO(2mL)溶剂中，先后加入铜(127mg,2mmol)、五水硫酸铜(13mg,0.05mmol)和1,5-已二炔(26μL,0.27mmol)。避光反应5小时后，加入乙酸乙酯(10mL)，过滤得到滤液。滤液用水洗3次(3×10mL)，饱和食盐水洗，无水硫酸钠干燥，过滤和减压浓缩得到粗产物。所得粗产物经硅胶柱色谱分离纯化，得到白色固体化合物1(总产率为51％)。
1H NMR(300MHz,CDCl3)δ7.55(s,2H),5.68(s,2H),4.70(t,J＝8.3Hz,2H),3.34(d,J＝6.1Hz,2H),3.20(d,J＝7.4Hz,4H),3.04(s,5H),2.79(dt,J＝11.0,9.6Hz,4H),2.35(d,J＝13.5Hz,2H),1.64–1.48(m,4H),1.39–1.11(m,10H)；13C NMR(75MHz,CDCl3)δ172.3,169.9,147.1,119.2,111.8,90.1,64.5,63.3,60.6,49.5,35.8,29.2,25.9,25.4,23.9,21.2；HR-ESI-MS m/z 599.3086[M+H]+。
实施例3化合物2的制备
Figure PCTCN2022140449-appb-000013
参照实施例2的合成方法，以叠氮化合物N10b为起始原料，得到白色固体化合物2(产率为47％)。
1H NMR(300MHz,CDCl3)δ7.30(d,J＝10.2Hz,2H),5.78(d,J＝22.4Hz,2H),4.81(s,2H),3.58(s,2H),3.38(s,4H),3.03(s,9H),2.43(dd,J＝11.1,5.7Hz,2H),1.86(s,2H),1.70–1.13(m,13H)；13C NMR(75MHz,CDCl3)δ172.3,171.1,146.9,120.4,111.5,90.4,64.1,60.1,48.7,32.3,28.1,25.9,25.0,23.9,21.7；HR-ESI-MS m/z 599.3088[M+H]+。
实施例4化合物3的制备
Figure PCTCN2022140449-appb-000014
参照实施例2的合成方法，以叠氮化合物N10a为起始原料与1,6-庚二炔反应，得到白色固体化合物3(产率为69％)。
1H NMR(300MHz,CDCl3)δ7.55(s,2H),5.71(s,2H),5.26(d,J＝5.7Hz,1H),4.73(t,J＝8.2Hz,2H),3.40(d,J＝6.0Hz,2H),3.18(dd,J＝32.4,6.3Hz,6H),2.93–2.67(m,8H),2.45(d,J＝13.4Hz,2H),2.08–1.95(m,2H),1.61(t,J＝9.5Hz,4H),1.44–1.11(m,10H),0.82(s,1H)；13C NMR(75MHz,CDCl3)δ172.2,170.0,147.4,119.0,111.6,90.2,64.8,63.2,61.1,49.9,36.3,29.2,26.4,25.1,23.6,21.1；HR-ESI-MS m/z 613.3245[M+H]+。
实施例5化合物4的制备
Figure PCTCN2022140449-appb-000015
参照实施例2的合成方法，以叠氮化合物N10b为起始原料与1,6-庚二炔反应，得到白色固体化合物4(产率为54％)。
1H NMR(300MHz,CDCl3)δ7.28(d,J＝8.8Hz,2H),5.83(s,2H),5.28(s,1H),4.78(d,J＝27.5Hz,2H),3.60(s,2H),3.42(d,J＝13.5Hz,4H),3.03(s,6H),2.70(t,J＝7.4Hz,4H),2.47(dd,J＝11.3,5.9Hz,2H),2.04–1.94(m,2H),1.88(s,1H),1.61(s,2H),1.50–1.41(m,5H),1.38(d,J＝8.5Hz,4H),1.23(s,1H),0.85(t,J＝6.7Hz,1H)；13C NMR(75MHz,CDCl3)δ172.5,171.2,147.6,120.1,111.5,90.2,64.2,60.1,48.7,32.5,28.8,28.2,26.1,24.7,24.0,21.7；HR-ESI-MS m/z 613.3245[M+H]+。
实施例6化合物5的制备
Figure PCTCN2022140449-appb-000016
参照实施例2的合成方法，以叠氮化合物N10a为起始原料与1,7-辛二炔反应，得到白色固体化合物5(产率为75％)。
1H NMR(300MHz,CDCl3)δ7.50(s,2H),5.71(s,2H),4.73(t,J＝8.3Hz,2H),3.40(d,J＝5.9Hz,2H),3.32–3.06(m,6H),2.90–2.78(m,3H),2.70(s,4H),2.45(d,J＝13.5Hz,2H),1.75–1.55(m,8H),1.41–1.16(m,9H),0.84(d,J＝5.6Hz,1H).13C NMR(75MHz,CDCl3)δ172.5,169.9,148.4,118.6,112.1,90.4,64.7,63.8,61.0,49.9,36.3,29.3,28.9,26.0,25.4,23.9,21.3；HR-ESI-MS m/z627.3390[M+H]+。
实施例7化合物6的制备
Figure PCTCN2022140449-appb-000017
参照实施例2的合成方法，以叠氮化合物N10b为起始原料与1,7-辛二炔反应，得到白色固体化合物6(产率为60％)。
1H NMR(300MHz,CDCl3)δ5.78(s,2H),5.24(s,1H),4.77(s,2H),3.55(s,2H),3.43–3.21(m,4H),2.98(s,6H),2.62(s,4H),2.40(dd,J＝10.6,5.6Hz,2H),1.82(s,2H),1.60(d,J＝17.4Hz,4H),1.52(s,2H),1.46–1.13(m,11H)；13C NMR(75MHz,CDCl3)δ172.2,171.0,147.6,119.8,111.3,90.3,63.9,60.2,60.0,48.5,32.2,28.4,27.9,26.0,25.0,23.8,21.5；HR-ESI-MS m/z 627.3382[M+H]+。
实施例8化合物7的制备
Figure PCTCN2022140449-appb-000018
参照实施例2的合成方法，以叠氮化合物N10a为起始原料与炔丙基醚反应，得到白色固体化合物7(产率为57％)。
1H NMR(300MHz,CDCl3)δ7.76(s,2H),5.62(s,2H),4.77–4.40(m,6H),3.40–3.00(m,8H),2.80–2.70(m,4H),2.35(s,2H),1.55–1.45(m,4H),1.30–1.00(m,10H)；13C NMR(75MHz,CDCl3)δ171.9,169.7,144.1,120.7,111.3,89.8,64.2,63.2,63.0,60.4,53.4,49.2,35.5,28.9,25.6,23.5,20.9；HR-ESI-MS m/z 615.3038[M+H]+。
实施例9化合物8的制备
Figure PCTCN2022140449-appb-000019
参照实施例2的合成方法，以叠氮化合物N10b为起始原料与炔丙基醚反应，得到白色固体化合物8(产率为57％)。
1H NMR(300MHz,CDCl3)δ7.58(d,J＝3.8Hz,2H),5.80(s,2H),5.25(s,1H),4.90–4.55(m,6H),3.65–3.27(m,6H),3.01(s,6H),2.43(dd,J＝10.6,5.5Hz,2H),1.48–1.24(m,11H)；13C NMR(75MHz,CDCl3)δ172.2,171.0,144.5,122.1,111.5,90.4,64.0,63.4,48.6,32.3,29.6,28.0,25.9,23.8,21.4.；HR-ESI-MS m/z 615.3038[M+H]+。
实施例10化合物9的制备
Figure PCTCN2022140449-appb-000020
参照实施例2的合成方法，以叠氮化合物N10a为起始原料与化合物N4反应，得到白色固体化合物9(产率为56％)。
1H NMR(300MHz,CDCl3)7.67(s,1H),5.74(s,1H),5.58(s,1H),4.77(t,J＝8.4Hz,1H),3.89–3.78(m,2H),3.45(d,J＝6.5Hz,1H),3.33–3.20(m,3H),3.18–3.09(m,2H),2.92–2.81(m,6H),2.61(d,J＝16.4Hz,1H),2.48(d,J＝10.9Hz,2H),2.14(d,J＝1.1Hz,1H),1.92(d,J＝10.5Hz,2H),2.00–1.70(m,3H),1.65(d,J＝11.1Hz,1H),1.63–1.53(m,2H),1.43–1.22(m,8H)；13C NMR(75MHz,CDCl3)174.2,173.0,172.2,169.7,146.6,119.6,111.8,111.0,91.3,90.1,64.6,63.6,62.8,60.8,59.8,58.9,49.7,48.7,42.7,36.0,32.3,30.1,29.4,26.0,25.7,23.9,23.7,21.4,21.2；HR-ESI-MS m/z 533.2873[M+H]+。
实施例11化合物10的制备
Figure PCTCN2022140449-appb-000021
参照实施例2的合成方法，以叠氮化合物N10b为起始原料与化合物N4反应，得到白色固体化合物10(产率为64％)。
1H NMR(300MHz,CDCl3)7.42(s,1H),5.82(s,1H),5.58(s,1H),4.85–4.80(m,1H),3.86–3.74(m,2H),3.61–3.58(m,1H),3.39(d,J＝3.4Hz,1H),3.26–3.23(m,1H),3.12–3.00(m,4H),2.92–2.80(m,4H),2.64–2.60(m,3H),2.50–2.43(m,1H),2.15(s,1H),1.92–1.85(m,4H),1.59–1.28(m,10H)；13C NMR(75MHz,CDCl3)174.2,173.1,172.3,171.0,146.5,120.7,111.6,111.0,91.3,90.5,64.1,62.8,60.6,60.1,59.8,59.2,48.8,48.7,42.6,41.0,32.5,32.4,30.1,28.1,26.1,25.8,24.0,23.8,22.6,21.7,21.5；HR-ESI-MS m/z 533.2865[M+H]+。
实施例12新型一叶萩碱二聚体类化合物促神经分化作用
(1)实验方法：Neuro-2a细胞(购于American type culture collection细胞库)复苏后，以生长培养基(MEM+10％FBS+100U/mL的青霉素和100μg/mL的链霉素)培养，种植在100mm的培养皿中，放入37℃、含5％CO2的恒温培养箱培养。细胞长至60～70％进行传代，吸去培养皿中的培养液，加入适量PBS清洗后用0.25％的胰蛋白酶消化45s，待贴壁细胞呈圆球状后加生长培养基终止消化，混匀按1:10传代，三天一传。诱导神经细胞株分化时，细胞种植密度为2×104个/35mm培养皿或1×104个/孔(12孔板)，以生长培养基培养24h后换为分化培养基(MEM+0.5％FBS+100U/mL的青霉素和100μg/mL的链霉素)，并加入浓度为25μM的一叶萩碱二聚体类化合物处理48h。神经细胞株的分化形态和神经突起通过免疫荧光染色的方法观察，具体步骤如下：
①用PBS(含Ca2+/Mg2+)洗细胞1次后，加入800μL 4％多聚甲醛/4％蔗糖，室温固定20～30min；
②用PBS洗3次，加入封闭液(含1％的牛血清白蛋白、4％的羊血清白蛋白和0.4％的Triton X-100的PBS)封闭20min；
③弃掉封闭液，加入含β-tubulin III抗体(1:3000)的一抗稀释液(含1％的牛血清白蛋白、1％的羊血清白蛋白和0.4％的Triton X-100的PBS)，室温孵育1h或4℃孵育过夜；
④用PBS洗3次，加入含荧光二抗Alexa Fluor-488goat anti-mouse IgG抗体(1:5000)的稀释液(含1％的牛血清白蛋白和0.4％的Triton X-100的PBS)，室温孵育1h；
⑤用PBS洗3次，进行封片。
采用正置荧光显微镜拍照，并利用Metamorph软件统计分析。定义神经突起长度大于20μm的细胞为分化的神经细胞。主要测量分析的参数有两个：1)细胞分化率；2)每个分化细胞的总神经突起长度。
实验结果：如表1所示，三氮唑为连接基团的化合物1～10在25μM时均具有促神经分化活性；其中，三氮唑化合物3在促神经细胞分化和促进神经突起生长两方面的活性均显著优于简单的一叶萩碱二聚体类化合物SN3-L6。如表2所示，化合物3在低剂量(1μM)时仍具有促神经分化活性，而此浓度下SN3-L6已经无活性。因此，化合物3较简单的一叶萩碱二聚体类化合物SN3-L6显示出更强的促进神经分化作用。
表1新型一叶萩碱二聚体类化合物在25μM剂量下促进Neuro-2a细胞分化的活性
Figure PCTCN2022140449-appb-000022
Figure PCTCN2022140449-appb-000023
实验结果的数据用mean±S.E.M(standard error of the mean)表示，设有三次独立的实验，每次每组至少分析100个细胞，并利用unpaired t-test检验法进行统计学处理，*P<0.05,**P<0.01,***P<0.001。
表2化合物3在1μM剂量下促进Neuro-2a细胞分化的活性
Figure PCTCN2022140449-appb-000024
实验结果的数据用mean±S.E.M(standard error of the mean)表示，设有三次独立的实验，每次每组至少分析100个细胞，并利用unpaired t-test检验法进行统计学处理，*P<0.05,**P<0.01。
实施例13新型一叶萩碱二聚体类化合物促神经细胞新生蛋白质合成作用
实验方法：用嘌呤霉素(puromycin)标记方法测定一叶萩碱二聚体类化合物处理后的神经细胞新生蛋白的合成量。将Neuro-2a细胞按6×105个/35mm培养皿的密度种植于35mm培养皿中，在37℃、含5％CO2的恒温培养箱培养过夜后，加入一叶萩碱二聚体类化合物混匀，使其终浓度为25μM，对照组加入等体积的DMSO，在37℃、含5％CO2的恒温培养箱中培养1h。再向培养基中加入1μM嘌呤霉素，继续在37℃、含5％CO2的恒温培养箱中培养0.5h。接着，用冰D-PBS洗两次，再用RIPA裂解液(含蛋白酶抑制剂)收集细胞裂解蛋白，随 后进行Western blot实验，用嘌呤霉素的抗体检测嘌呤霉素标记的新生蛋白含量。
实验结果：如图1所示，化合物1～10在25μM剂量下均有明显的促进神经细胞蛋白质合成的作用。如图2所示，化合物3在10μM和1μM低剂量下仍具有促进蛋白质合成的作用，而简单的一叶萩碱二聚体类化合物SN3-L6在1μM时无活性，说明化合物3较简单的一叶萩碱二聚体类化合物SN3-L6显示出更强的促进神经细胞蛋白质合成的作用。
所述图1为Western blot检测嘌呤霉素标记新生蛋白质含量的柱状统计图。Neuro-2a细胞经25μM化合物处理1h后，再加入1μM嘌呤霉素处理0.5h，设DMSO组为对照组。实验结果的数据用mean±S.E.M(standard error of the mean)表示，设有三次独立的实验，并用One-way ANOVA检验法进行统计学处理，**P<0.01,***P<0.001。
所述图2为Western blot检测嘌呤霉素标记新生蛋白质含量的柱状统计图。Neuro-2a细胞经1μM或10μM化合物处理1h后，再加入1μM嘌呤霉素处理0.5h，设DMSO组为对照组。实验结果的数据用mean±S.E.M(standard error of the mean)表示，设有三次独立的实验，并用One-way ANOVA检验法进行统计学处理，*P<0.05,different compounds vs DMSO。
实施例14化合物3的细胞内富集能力测定
前期研究发现，简单的一叶萩碱二聚体类化合物SN3-L6的体内代谢较快，因此我们首先对三氮唑化合物3进行了细胞内富集能力的测试，并与SN3-L6进行比较。
(1)实验方法：在35mm培养皿种植6×105个Neuro-2a细胞，放于37℃、含5％CO2的恒温培养箱过夜后，在其生长培养基中加入25μM的化合物3和SN3-L6，具体步骤如下：
①加入化合物孵育1h和24h后，用冰D-PBS将培养皿洗3次，在每个培养皿中加入396μL生理盐水，刮取细胞并转移至1.5mL EP管中；
②冰上超声裂解30min后，加入4μL内标化合物，使其终浓度为50ng/mL；
③加入600μL乙酸乙酯(分析纯)涡旋10min，室温静置10min待两液相分层，再用10000rpm 4℃离心10min，取上层有机相500μL，重复萃取3次；
④通过真空离心浓缩仪在30℃挥干有机萃取溶剂；
⑤在样品中加入400μL 80％甲醇复溶，室温水浴超声30min使其完全溶解，再涡旋2min；
⑥10000rpm室温离心10min，再取100μL上清溶液至液相分析瓶中，使用高分辨液相质谱联用仪进行含量检测。
根据公式f′＝fi/fs＝(Mi/Ai)/(Ms/As)与(Ai/As)*f′＝Ci/Cs以及回收率公式计算出细胞内化合物的含量。再通过Graph prism 5.0软件拟合出不同作用时间的细胞内化合物浓度折线图。
实验结果：如表3所示，孵育1h和24h后，Neuro-2a细胞中的化合物3的含量高于SN3-L6，说明化合物3和13比简单的一叶萩碱二聚体类化合物SN3-L6更好地进入细胞并在细胞内富集。
表3化合物3作用不同时间后在Neuro-2a细胞中的含量测定
Figure PCTCN2022140449-appb-000025
实验结果的数据用mean±S.E.M(standard error of the mean)表示，设有三组平行样品。
实施例15化合物3的体内代谢分析
根据实施例21～23的实验结果，化合物3活性最优、进入细胞能力最强，因此我们选择化合 物3为代表化合物来研究其在动物体内的代谢情况。
实验方法：将化合物3或SN3-L6以25mg/kg的剂量灌胃给药C57BL/6小鼠，分别在给药后5min、30min、60min和120min共4个时间点进行眼球取血，并收取脑组织，每组各3只鼠。具体步骤如下：
小鼠腹腔注射戊巴比妥钠，待其麻醉完全后，摘眼球取血，收集于含有抗凝剂的1.5mL离心管中，轻轻颠倒混匀。取500μL血液；4℃离心3800rpm，20min；取上层血浆300μL，加入1mL提取剂(乙酸乙酯)，涡旋5min；
用PBS灌注小鼠后取出全脑，称重，加入200μL去离子水，在冰上匀浆后，加入1mL乙酸乙酯，涡旋5min；
血液和脑组织分别加入提取剂涡旋后，常温静置15～30min，4℃，12000rpm离心20min；取上层有机相，保留到新管中；对下层匀浆液再用相同的方法抽提2次；将两次的抽提物混合后，置于氮吹仪中挥干；用100～200μL的流动相(甲醇或乙腈)进行溶解，使用高分辨液相质谱联用仪进行含量检测。
实验结果：如表4所示，在5min、30min、60min和120min时，化合物3在血浆与脑组织中的含量均明显高于简单的一叶萩碱二聚体类化合物SN3-L6，说明化合物3在小鼠血液中的代谢稳定性明显优于SN3-L6。
表4化合物3和SN3-L6灌胃给药不同时间后在小鼠血浆中的含量测定
Figure PCTCN2022140449-appb-000026
实验结果的数据用mean±S.E.M(standard error of the mean)表示，每组3只小鼠；
表5化合物3和SN3-L6灌胃给药不同时间后在小鼠脑组织中的含量测定
Figure PCTCN2022140449-appb-000027
实验结果的数据用mean±S.E.M(standard error of the mean)表示，每组3只小鼠；BLQ:Below limit of Quantitation。
实施例16化合物3改善AD模型小鼠在新物体识别测试中的记忆认知功能
实验方法：新物体识别测试是利用啮齿类动物先天对新物体有探索倾向的原理而建立的学习记忆测试方法，衡量动物短时间或长时间的记忆、认知功能。本实施例使用APP1/PS1双转基因小鼠，该小鼠是经典AD模型鼠，在4～5月龄时开始出现认知障碍，并逐渐加重。实验使用雄性APP1/PS1小鼠，随机分组(每组10只鼠)，进行化合物3、SN3-L6或溶剂给药，在7月龄时进行灌胃给药，每日一次，持续4周，化合物3和SN3-L6均设置三个给药剂量(6.25mg/kg、12.5mg/kg和25mg/kg)，溶剂为生理盐水(加入5％吐温-80助溶)，每只小鼠按10μL/g灌胃相应体积的药物或溶剂。此外，设置一组同年龄雄性野生(WT)小鼠，给与溶剂。给药结束后进行新物体识别测试，实验主要分为适应阶段、训练阶段和测试阶段：
适应阶段：将小鼠依次放入没有任何物体的实验装置(40×40×40cm白色塑料箱)中，使其自由探索5min以适应实验环境，减少动物探索新环境对实验的干扰；
训练阶段：在实验装置底板上用双面胶粘上两个完全相同的物体(白色塑料瓶盖，直径4cm，高2.8cm)，将小鼠以背对两个物体的方式放入实验装置中，使其自由探索10min并用视频系统记录，结束后将动物取出放回饲养笼中。间隔1h后，进行测试期实验；
测试阶段：将其中一个物体换成不同形状的物体(方形积木，4.6×2.2×3.5cm)，原有物体称之为旧物体或熟悉物体，新换物体称之为新物体或新奇物体，将小鼠以背对两个物体的方式放入实验装置中，使其自由探索10min并用视频系统记录，结束后将动物取出放回饲养笼中；
用Topscan 3.0软件分析小鼠运动总距离与嗅探不同物体的时间。
实验结果：如图3所示，溶剂组中，WT小鼠对新旧物体的嗅探时间有显著性差异，而AD模型小鼠对新旧物体的嗅探时间无差异，说明AD小鼠的记忆认知功能受损；连续4周灌胃12.5或25mg/kg SN3-L6或化合物3后，AD小鼠对新旧物体的嗅探时间呈现显著区别；然而，在6.25mg/kg给药剂量时，化合物3仍可有效恢复AD小鼠的认知水平，SN3-L6则没有作用。这说明相比SN3-L6，化合物3的有效剂量更低，可以更佳地改善AD模型小鼠在新物体识别测试中的记忆认知功能。
所述图3为不同组小鼠在测试阶段表现出的新物体识别能力。new为嗅探新物体时间，old为嗅探旧物体时间，recognition index＝嗅探新或旧物体时间/嗅探新旧物体的总时间。每组各12只鼠，实验结果的数据用mean±S.E.M(standard error of the mean)表示，用One-way ANOVA检验法进行统计学处理，*P<0.05,**P<0.01,****P<0.0001,ns,no significance。
实施例17化合物3改善AD模型小鼠在Morris水迷宫测试中的空间学习记忆功能
实验方法：Morris水迷宫测试通过让啮齿类动物在水中寻找平台，并分析其寻找平台所用时间和所走路径来判断其空间学习记忆功能。实验对象、分组方案和给药方式与实施例16一致。实验过程主要分为训练阶段和测试阶段：
训练阶段：在圆形水缸(直径120cm，深度45cm)中注入含有二氧化钛(降低水下能见度)的水，温度保持在25℃。指定两个互相垂直的主轴，将池划分为四个相等的象限，并将这些轴线与池边缘的交点指定为北(N)、南(S)、东(E)和西(W)。将一个圆形平台(直径10cm，高30cm)放置在某一象限(目标象限)的中心，在不同象限对应的墙上贴上不同图案，作为空间学习的迷宫线索。训练共持续7天，每天训练3次(每组90s)，每次训练间隔30min。第1天时将圆形平台置于水面上。实验时，将小鼠面向池壁放入水中，3次训练时放入的位置均不同(N、NE和E)，如果在90s内未找到平台，需将其引至平台，这时潜伏期记为90s，并用视频系统记录实验过程。在第2～7天，测试与第1天的步骤相同，只是平台被淹没在水面以下0.5cm处；
测试阶段：在第8天(最后一次试验后24小时)时撤去平台，将小鼠面向池壁放入水中，用 视频系统记录其90s内的运动情况；
采用Topscan 3.0软件分析小鼠在不同象限的停留时间与游泳速度。
实验结果：如图4所示，连续4周灌胃给药后，溶剂对照组的AD小鼠在目标象限和其他象限的停留时间无差异，说明AD小鼠在Morris水迷宫测试中的空间学习记忆功能受损；给与25mg/kg SN3-L6或化合物3后，AD小鼠在目标象限停留时间最长，与其他象限的停留时间显示出统计差异；然而，在6.25或12.5mg/kg给药剂量时，化合物3可有效恢复AD小鼠的认知水平，SN3-L6则没有作用。说明化合物3的有效剂量更低，可以更佳的改善AD小鼠在Morris水迷宫测试中的空间学习记忆功能。
所述图4为不同组小鼠在测试阶段分别在目标象限和其他象限的停留时间。Target为小鼠在目标象限的停留时间，Average of others为小鼠在其他象限的停留时间。每组各12只小鼠，实验结果的数据用mean±S.E.M(standard error of the mean)表示，用One-way ANOVA检验法进行统计学处理，*P<0.05,**P<0.01,***P<0.001,****P<0.0001,ns,no significance。
需要说明的是，以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。

Claims (9)

  1. 一种具有式I所示结构的新型一叶萩碱二聚体化合物或其药学上可接受的盐或其立体异构体或其前药分子：
    Figure PCTCN2022140449-appb-100001
    式中：
    连接基团Linker选自：
    Figure PCTCN2022140449-appb-100002
    X选自
    Figure PCTCN2022140449-appb-100003
    或者
    Figure PCTCN2022140449-appb-100004
    n为1、2、3或4。
  2. 根据权利要求1所述的新型一叶萩碱二聚体，其特征在于，所述Linker＝A时，所述的二聚体类化合物具有式II所示的结构式或其药学上可接受的盐或其立体异构体或其前药分子：
    Figure PCTCN2022140449-appb-100005
    其中：X选自
    Figure PCTCN2022140449-appb-100006
    或者
    Figure PCTCN2022140449-appb-100007
    n为1、2、3或4。
  3. 根据权利要求2所述的新型一叶萩碱二聚体，所述二聚体类化合物选自：
    Figure PCTCN2022140449-appb-100008
    Figure PCTCN2022140449-appb-100009
  4. 根据权利要求1所述的新型一叶萩碱二聚体，其特征在于，所述Linker＝B时，所述的二聚体类化合物具有通式III所示的结构式或其药学上可接受的盐或其立体异构体或其前药分子：
    Figure PCTCN2022140449-appb-100010
  5. 根据权利要求4所述的新型一叶萩碱二聚体，所述二聚体类化合物选自：
    Figure PCTCN2022140449-appb-100011
  6. 一种权利要求2所述的通式II所示的一叶萩碱二聚体化合物的制备方法，其特征在于，该方法包括：
    Figure PCTCN2022140449-appb-100012
    常温下，将一叶萩碱(1.0equiv.)溶于搅拌的二氯甲烷溶剂中，先后加入三甲基硅基叠氮(5.0equiv.)、醋酸(5.0equiv.)和DBU(0.05equiv.)；升温至35℃后，反应8小时，有机溶剂经减压浓缩得到粗产物；所得粗产物经硅胶柱色谱分离纯化，得到一对非对映异构体叠氮化合物N10a和N10b；随后，将叠氮化合物N10a或N10b(1.0equiv.)溶于搅拌的DMSO溶剂中，先 后加入铜(2equiv.)、五水硫酸铜(0.05equiv.)和二炔类化合物(0.27equiv.)；避光反应5小时后，加入乙酸乙酯，过滤得到滤液；滤液用水洗3次，饱和食盐水洗，无水硫酸钠干燥，过滤和减压浓缩得到粗产物；所得粗产物经硅胶柱色谱分离纯化，得到通式化合物II；
    X如权利要求1中所定义。
  7. 一种权利要求4所述的通式III所示的一叶萩碱二聚体化合物的制备方法，其特征在于，该方法包括：
    Figure PCTCN2022140449-appb-100013
    将叠氮化合物N10a或N10b(1.0equiv.)溶于搅拌的DMSO溶剂中，先后加入铜(2.0equiv.)、五水硫酸铜(0.05equiv.)和通式化合物III-1(0.27equiv.)；避光反应5小时后，加入乙酸乙酯，过滤得到滤液；滤液用水洗3次，饱和食盐水洗，无水硫酸钠干燥，过滤和减压浓缩得到粗产物；所得粗产物经硅胶柱色谱分离纯化，得到通式化合物III。
  8. 一种药物组合物，其特征在于，其包括权利要求1～7中任一项所述的通式I、II和III所示的一叶萩碱二聚体，或其药学上可接受的盐或其立体异构体或其前药分子，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
  9. 根据权利要求1～7中任一项所述的通式I、II和III所示的一叶萩碱二聚体，或其药学上可接受的盐或其立体异构体或其前药分子、或根据权利要求8所述的药物组合物在制备用于治疗阿尔茨海默病药物中的应用。
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