WO2021238328A1 - 一种含有异羟肟酸的化合物及其制备方法、应用 - Google Patents

一种含有异羟肟酸的化合物及其制备方法、应用 Download PDF

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WO2021238328A1
WO2021238328A1 PCT/CN2021/079482 CN2021079482W WO2021238328A1 WO 2021238328 A1 WO2021238328 A1 WO 2021238328A1 CN 2021079482 W CN2021079482 W CN 2021079482W WO 2021238328 A1 WO2021238328 A1 WO 2021238328A1
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alkyl
unsaturated
saturated
compound
phenyl
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French (fr)
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王进欣
杨侃
陈瑜
余荩颖
董继斌
顾勤兰
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中国药科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61P25/24—Antidepressants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/50—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D333/52—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes
    • C07D333/54—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
    • C07D333/56—Radicals substituted by oxygen atoms

Definitions

  • the invention relates to a hydroxamic acid structure compound, in particular to a compound containing hydroxamic acid and a preparation method and application thereof.
  • Acid sphingomyelinase (ASM) hydrolysis of sphingomyelin is the fastest and most direct way to generate ceramide in the body. So far, a variety of endogenous and exogenous factors have been found, including tumor necrosis factor- ⁇ (TNF- ⁇ ), interleukin- ⁇ (IL- ⁇ ), interferon- ⁇ , etc., as well as oxidative stress and ionizing radiation. , Ultraviolet radiation, thermal shock, trauma, bacterial infection and chemical reagents can all activate acid sphingomyelinase, leading to the massive production and accumulation of ceramide. After the level of ceramide increases, it participates in signal transport and material transfer inside and outside the cell (FEBS Lett, 2010, 584(9): 1728-1740).
  • Acid sphingomyelinase is a potential drug target, and there is an urgent need to develop new direct inhibitors of acid sphingomyelinase as candidate drugs for the treatment of related diseases.
  • the present invention finds for the first time that a compound containing hydroxamic acid is prepared, which is a new compound and an acid sphingomyelinase inhibitor at the same time.
  • the present invention is a compound containing hydroxamic acid and its preparation method and application, which is a novel acid sphingomyelinase inhibitor.
  • R 1 is C 1 -C 10 saturated alkyl straight chain, C 1 -C 10 unsaturated unsaturated straight chain alkyl, C 3- C 10 saturated branched chain alkyl, C 3 -C 10 unsaturated branched chain Alkyl group, C 3 -C 10 cycloalkyl group or phenyl group;
  • X is C, N, O or S
  • C n H m is C 1 -C 4 linear saturated alkyl, C 1 -C 4 unsaturated linear alkyl, C 1- C 4 saturated branched alkyl, C 1- C 4 unsaturated Branched alkyl group; wherein, 1 ⁇ n ⁇ 4, 2 ⁇ m ⁇ 8.
  • R 2 is a mono- or di-substituted phenyl group, wherein the substituents are respectively located at the ortho, meta or para positions of the benzene ring, and the substituents can specifically be hydrogen, fluorine, chlorine, bromine, cyano, OR 3 , CF 3 , SF 5 ;
  • R 2 is pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, C 1 -C 10 linear saturated alkyl, C 1 -C 10 unsaturated linear alkyl, saturated C 3 -C 10 branched alkyl or unsaturated C 3 -C 10 branched alkyl; wherein oR R 3 3 is H, C linear or branched alkyl group of 1 -C 4, C 3 - C 6 cycloalkyl, phenyl;
  • W, Y or Z is any one of the four elements above C, N, O, and S.
  • R 4 , R 5 or R 6 are H, F, Cl, Br, CF 3 , CHF 2 , COCF 3 , COCH 3 , COC 2 H 5 , CN; C 1 -C 6 alkoxy, C1-C6 saturated Any one of linear alkyl, C 1 -C 6 unsaturated linear alkyl, C 3 -C 6 saturated branched alkyl, C 3 -C 6 unsaturated branched alkyl, and C3-C6 cycloalkane kind.
  • R 1 is phenyl, n-butyl or n-propyl
  • R 2 is selected as 4-chlorophenyl, 4-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-chloro-3-trifluoromethyl-phenyl or 2,4-bis(trifluoromethyl)-phenyl
  • X is C or O
  • W or Z is N
  • Y is C
  • R 4 , R 5 , R 6 is selected as H.
  • the compound is prepared for the treatment of atherosclerosis, diabetes, emphysema, pulmonary edema, pulmonary fibrosis, COPD, pulmonary hypertension, cystic fibrosis, non-alcoholic fatty liver, Alzheimer, Application of drugs for multiple sclerosis, stroke or depression.
  • the present invention first discovered a new compound such as (I1-I8), which is a new skeleton, and the efficacy data show that the above-mentioned compound is an acid sphingomyelinase inhibitor; and further pharmacodynamic studies have been done on the compound I-1, and the compound I-1 has Antidepressant activity and anti-atherosclerotic activity have good clinical prospects.
  • Figure 1 is the open-box experimental study of compound I-1 in Figure 1, where AD is the time spent in the center grid, the number of grid passes, the number of uprights, and the number of combing; *, vs Control group, p ⁇ 0.01; #, vs Model group ,p ⁇ 0.01;
  • Figure 4 A view of the aortic arch plaque, where A is the control group 1, B is the control group 2, C is the low-dose group and D is the high-dose group;
  • Figure 5 is a statistical analysis diagram of aortic oil red O staining plaque volume
  • Figure 6 is an observation picture of aortic oil red O stained plaque; where A is the control group 1, B is the control group 2, C is the low-dose group and D is the high-dose group;
  • Figure 7 shows the effect of compound I-1 on plasma lipid content, where A is total triglycerides, B is total cholesterol, C is high-density lipoprotein, and D is low-density lipoprotein.
  • the first step is to dissolve 168mg (0.41mmol) of the raw material in 4mL of methanol, add 4mL of H 2 O, 82mg (2.05mmol) of NaOH, reflux at 80°C, react for 2 hours, TLC monitors the completion of the reaction, add 20mL of 10% HCl, white precipitation
  • the solid products 7a and 7a were dried and dissolved in 10 mL of dried anhydrous dichloromethane, and 0.1 mL of thionyl chloride was added to the reaction flask with a pipette, protected by nitrogen, and condensed to reflux at 45°C.
  • the pH was adjusted with 10% dilute HCl to adjust the pH of the solution to neutral, and a large amount of white solid was precipitated. After refrigerating for a period of time, suction filtration was used to obtain 98 mg of white solid product with a yield of 61%.
  • Example 2 Compounds inhibit acid sphingomyelinase activity test
  • Acid sphingomyelinase can hydrolyze sphingomyelin in the cell to generate ceramide.
  • fluorescently labeled reaction substrate Al, USA
  • different enzyme activities catalyze the production of different amounts of products.
  • the enzyme can be inspected by detecting the content of the product.
  • the present invention performs experimental design based on this principle.
  • Extract the protein in the cultured cells add buffer, fluorescently labeled reaction substrate, then add different concentrations of compounds, set up a blank control group, perform fluorescence analysis after the reaction, and finally calculate the IC50 value of the compound.
  • the present invention establishes a rat chronic depression model through reserpine (reference document: NeurotoxRes, 2014, doi: 10.1007/s12640-013-9454-8).
  • the specific experimental steps are as follows:
  • Rats in the remaining groups were injected with reserpine 0.2 mg/Kg intraperitoneally, once a day for three days.
  • the other groups were injected intraperitoneally with different doses of compound I-1 (3mg/Kg, 6mg/Kg, 12mg/Kg) or the positive drug amitriptyline (6mg/Kg)/or no drugs were given.
  • the open-box experiment was carried out in sequence (reference: Chinese Pharmacy, 2016, 27(19): 2697-2699.), and the sugar water preference experiment was carried out after the experiment.
  • the open-box results showed that the administration of compound I-1 can significantly improve the staying time of the rat in the central grid, the number of grid passes, the number of standing up, and the number of grooming.
  • the behavioral indicators of high-dose (12mg/kg) I-1 rats in the open box were equivalent to the effect of amitriptyline (6mg/kg) in the positive control group.
  • the above data show that compound I-1 has significant antidepressant activity.
  • Apo E is an important component of plasma lipoproteins. It plays an important role in regulating plasma cholesterol levels. It is an important molecular target for the occurrence and development of hyperlipidemia and atherosclerosis. The development of AS lesions is very similar to that of humans. At present, ApoE knockout mice have become one of the most important animal models for studying the pathogenesis of AS and anti-AS pharmacology.
  • mice fed with a high-fat diet are commonly used animal models for the study of atherosclerosis, see (Chinese Journal of Integrative Medicine, 2019) , 25(02):108-115.).
  • mice were given high-fat feeding for 2 weeks, and 8 weeks after the right carotid artery cannulation, it was found that their TC, triacylglycerol (TG), low-density lipoprotein cholesterol (LDL-C), etc. were significantly increased. There is obvious atherosclerotic plaque formation at the cannula of the common carotid artery. (For anatomical methods, see Journal of Anatomy, 2018,41(01):16-19). The mice were randomly divided into 6 groups with 5 mice in each group. By intraperitoneal injection, each group was given blank solvent, blank solvent, 12 mg/Kg compound I-1, and 40 mg/Kg compound I-1. It is administered once a day for a total of 8 weeks. After the experiment, the animals were sacrificed and the aorta was separated.
  • TC triacylglycerol
  • LDL-C low-density lipoprotein cholesterol
  • Compound I-1 can significantly reduce the area of atherosclerotic plaques at 12mg/Kg and 40mg/Kg, but there is no quantitative dependence on the whole.

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Abstract

提供一种式I所示的含有异羟肟酸的化合物及其制备方法、应用。通过生物学实验进行了酸性鞘磷脂酶抑制活性评价；对I-1化合物进一步做了体内药效研究，显示I-1化合物具有显著的抗抑郁活性和抗动脉粥样硬化活性，为酸性鞘磷脂酶抑制剂的进一步开发提供了可行性。

Description

一种含有异羟肟酸的化合物及其制备方法、应用 技术领域
本发明涉及异羟肟酸结构化合物，特别涉及一种含有异羟肟酸的化合物及其制备方法、应用。
背景技术
酸性鞘磷脂酶(ASM)水解鞘磷脂是体内生成神经酰胺最快最直接的途径。迄今为止，已发现多种内源性和外源性的因子包括肿瘤坏死因子-α(TNF-α)、白介素-β(IL-β)、干扰素-γ等，以及氧化应激、离子辐射、紫外线照射、热撞击、创伤、细菌感染和化学试剂等均可以激活酸性鞘磷脂酶，导致神经酰胺的大量生成和聚集。神经酰胺水平升高后，参与细胞内外的信号转运和物质传递(FEBSLett,2010,584(9):1728-1740)。
大量研究表明，酸性鞘磷脂酶—神经酰胺通路参与体内炎症、细胞凋亡和氧化应激等过程，与多种疾病的发生发展密切相关(Progress in Lipid Research,2016,61:51–62；Apoptosis,2015,20:607–620)。目前已发现酸性鞘磷脂酶参与的疾病包括动脉粥样硬化(AS)，肺部纤维化及囊性纤维化(CF)，非酒精性脂肪肝，阿尔兹海默(AD)，多发性硬化症(MS)，抑郁症等(TheFASEBJournal,2008,22:3419-3431；Biol.Chem.2015,396:707–736)。
通过抑制酸性鞘磷脂酶，使神经酰胺恢复正常水平，能够有效地缓解相关疾病的病症。目前高效、特异性酸性鞘磷脂酶抑制剂非常缺乏，文献报道的少量酸性鞘磷脂酶直接抑制剂为底物类似物，二磷酸酯类，3,5-二磷酸肌醇类等，具有选择性差，类药性差、对磷酸酯酶稳定性差、透膜能力差等缺陷，不能应用到相关疾病的药物开发中(CellPhysiol.Biochem.2010,26:01-08)。
研究表明酸性鞘磷脂酶—神经酰胺通路直接参与动脉粥样硬化的病变进程。调控神经酰胺的代谢途径很可能是动脉粥样硬化的潜在治疗途径。目前治疗动脉粥样硬化的药物，存在副作用和疗效不佳的缺陷。发现新作用机制的治疗动脉粥样硬化的药物，寻找新的有效治疗靶点，开发新的临床疗效理想、副作用小的抗动脉粥样硬化药物具有重要的临床和科学研究意义。
目前一些伴非典型特征的抑郁症尚无优势的治疗药物，导致自残自杀的风险升高。同时临床抗抑郁药物起效慢，副作用大也是抗抑郁药面临的严重问题。研究表明酸性鞘磷脂酶的抑制和神经酰胺的降低在抑郁症疾病发展过程中起着关键作用，ASM抑制剂可以干扰鞘磷脂酶-神经酰胺途径介导的信号传导，因此，开发新靶标、新作用机制的新型抗抑郁药具有重要的临床意义和科学意义。
酸性鞘磷脂酶是一个潜在的药物靶标，目前亟需开发新型酸性鞘磷脂酶直接抑制剂作为治疗相关疾病的候选药物。
本发明首次发现制备获得一种含有异羟肟酸的化合物，其为新化合物，同时为酸性鞘磷脂酶抑制剂。
发明内容
发明目的：本发明一种含有异羟肟酸的化合物及其制备方法、应用，其是新型的酸性鞘磷脂酶抑制剂。
技术方案
一种含有异羟肟酸的化合物，其结构由式I所示:
Figure PCTCN2021079482-appb-000001
或式(I)化合物的药学上可接受的盐或前药；其中，式I中：
R 1为C 1-C 10的饱和烷基直链、C 1-C 10的不饱和不饱和直链烷基、C 3-C 10饱和支链烷基、C 3-C 10不饱和支链烷基、C 3-C 10环烷基或苯基；
X为C、N、O或S；
C nH m为C 1-C 4的饱和烷基直链、C 1-C 4的不饱和直链烷基、C 1-C 4的饱和支链烷基、C 1-C 4的不饱和支链烷基；其中，1≤n≤4,2≤m≤8。
R 2是单取代或双取代的苯基，其中取代基分别位于苯环的邻位、间位或对位，取代基具体可为氢、氟、氯、溴、氰基、OR 3、CF 3、SF 5；R 2为吡啶-2-基、吡啶-3-基、吡啶-4-基、C 1-C 10的饱和烷基直链、C 1-C 10的不饱和直链烷基、C 3-C 10饱和支链烷基或C 3-C 10不饱和支链烷基；其中OR 3中的R 3为H、C 1-C 4的直链或支链烷基、C 3-C 6的环烷基、苯基；
W、Y或Z为C、N、O、S以上四种元素的任意一种。
R 4、R 5或R 6为H、F、Cl、Br、CF 3、CHF 2、COCF 3、COCH 3,COC 2H 5,CN；C 1-C 6烷氧基、C1-C6的饱和烷基直链、C 1-C 6的不饱和直链烷基、C 3-C 6饱和支链烷基、C 3-C 6不饱和支链烷基及C3-C6环烷烃基中任意一种。
所述的化合物，其特征在于R 1为苯基、正丁基或正丙基；R 2选为4-氯苯基、4-三氟甲基苯基、3-三氟甲基苯基、4-氯-3-三氟甲基-苯基或2，4-双(三氟甲基)-苯基；X为C或O；W或Z为N；Y为C，R 4、R 5、R 6选为H。
所述的化合物，其特征在于所述化合物如下结构任意一种：
Figure PCTCN2021079482-appb-000002
所述化合物在制备酸性鞘磷脂酶抑制剂中的应用。
所述化合物在制备用于治疗动脉粥样硬化、糖尿病、肺气肿、肺水肿、肺部纤维化、慢阻肺、肺动脉高压、囊性纤维化、非酒精性脂肪肝、阿尔兹海默、多发性硬化症、脑卒中或抑郁症药物中的应用。
有益效果
本发明首先发现全新化合物如(I1-I8)，其是全新骨架，通过药效数据显示上述化 合物为酸性鞘磷脂酶抑制剂；并I-1化合物做了进一步药效研究，I-1化合物具有抗抑郁活性和抗动脉粥样硬化活性，具有良好临床前景。
附图说明
图1为图1化合物I-1敞箱实验研究，其中A-D依次为在中央格停留时间、方格穿行次数、直立次数和梳理次数；*，vs Control group,p<0.01；#，vs Model group,p<0.01；
图2化合物I-1糖水偏好实验研究；
图3化合物I-1降低动脉粥样硬化小鼠斑块面积；
图4为主动脉弓斑块体视图，其中A为对照组1，B为对照组2，C为低剂量组D为高剂量组；
图5为大动脉油红O染色斑块体积统计分析图；
图6为大动脉油红O染色斑块观察图片；其中A为对照组1，B为对照组2，C为低剂量组D为高剂量组；
图7为化合物I-1对血浆脂质含量影响，其中A为总甘油三酯，B为总胆固醇，C为高密度脂蛋白，D为低密度脂蛋白。
具体实施方式
实施例1化合物合成
1.3-((4-羟苯基)氨基)-4-硝基苯甲酸乙酯(3a)的制备
Figure PCTCN2021079482-appb-000003
将原料3-氟-4硝基苯甲酸1g(5.4mmol)溶于10mL乙醇中，向其缓慢滴加氯化亚砜(1.2mL，16.2mmol)，80℃冷凝回流。反应3h后，冷却至室温，旋蒸浓缩，向反应瓶中加入40mL饱和碳酸氢钠并用100mL乙酸乙酯分三次萃取，合并有机相，用无水硫酸钠干燥。抽滤，滤液旋干得到淡黄色的粗品中间体1。得到的中间体1溶于15mLN，N-二甲基甲酰胺(DMF)中，加入对羟基苯胺708mg(6.5mmol)，三乙胺1636mg，110℃冷凝回流，反应6h冷却至室温，向反应液加入10％的稀HCl溶液40mL，再加入100mL乙酸乙酯分三次萃取，合并有机相，无水硫酸钠干燥。柱层析纯化(石油醚：乙酸乙酯＝16:1)后得到红色固体1.2g(3a)，收率73.6％。
1H NMR(400MHz,DMSO-d6)δ9.64(s,1H),9.38(s,1H),8.21(dd,J＝8.9,3.1Hz,1H),7.49(d,J＝1.7Hz,1H),7.30–7.06(m,2H),7.00–6.72(m,1H),4.26(q,J＝7.1Hz,1H),1.24(t,J＝7.0Hz,3H),ESI-MS m/z：303.1[M+H] +
2.4-氨基-3-((4-羟基苯基)氨基)苯甲酸乙酯(4a)的制备
Figure PCTCN2021079482-appb-000004
将原料1.2g(3.97mmol)溶于15ml乙醇中，加入3mL乙酸，锌粉1578mg(23.82mmol)，室温搅拌12h，TLC检测反应完毕后抽滤，滤饼洗涤至没有荧光，滤液旋干得到灰绿色的粗品。粗品在PE/EA体系重结晶，得到灰白色的固体1g(4a)，收率92.6％。
1H NMR(400MHz,DMSO-d6)δ8.90(s,1H),7.44(d,J＝1.8Hz,1H),7.35(dd,J＝6.6,1.7Hz,1H),6.77–6.61(m,6H),5.55(s,2H),4.17(q,2H),1.23(t,J＝7.1Hz,3H),ESI-MS m/z：273.1[M+H] +
3.1-(4-羟苯基)-1H-苯并[d]咪唑-6-羧酸乙酯(5a)的制备
Figure PCTCN2021079482-appb-000005
将原料500mg(1.84mmol)溶于干燥的3.8g原甲酸三甲酯(36mmol)，110℃冷凝回流，反应4h，TLC检测反应完毕后，冷却至室温。旋蒸部分溶剂，加入40mL10％稀盐酸振荡5min，加入100mL乙酸乙酯分三次萃取，再用饱和氯化钠溶液洗涤两次，合并有机相，无水硫酸钠干燥。柱层析纯化(石油醚：乙酸乙酯＝4:1)得到淡棕色固体378mg(5a)，收率65.1％。 1H NMR(300MHz,DMSO-d 6)δ8.76(s,1H),8.12(dd,J＝7.5,1.6Hz,1H),8.04–7.99(m,2H),7.70(d,1H),7.61–7.56(m,2H),6.88–6.83(m,2H),4.33(q,2H),1.37(t,3H),ESI-MS m/z：283.1[M+H] +
4.1-(4-((4-氯苄基)氧基)苯基)-1H-苯并[d]咪唑-6-羧酸乙酯(6a)的制备
Figure PCTCN2021079482-appb-000006
将原料200mg(0.72mmol)和对氯溴苄176mg(0.86mmol)溶于10mL丙酮，加入702mg碳酸铯(2.16mmol),碘化钾催化量，65℃冷凝回流。反应12h后，TLC检测反应完毕，抽滤。柱层析纯化(石油醚：乙酸乙酯＝2:1)得到黄色固体168mg，收率59.6％。 1H NMR(300MHz,DMSO-d 6)δ8.73(s,1H),8.07(s,1H),7.93(q,J＝8.6Hz,2H),7.54(t,J＝7.5Hz,6H),7.32(d,J＝8.7Hz,2H),5.27(s,2H),4.36(q,J＝7.1Hz,2H),1.35(t,J＝7.0Hz,3H).ESI-MS m/z：407.1[M+H] +
5.1-(4-((4-氯苄基)氧基)苯基)-N-羟基-1H-苯并[d]咪唑-6-羧酰胺(I-1)的制备
Figure PCTCN2021079482-appb-000007
第一步先将168mg(0.41mmol)原料溶于4mL甲醇中，加入4mLH 2O，82mg(2.05mmol)NaOH，80℃冷凝回流，反应2h，TLC监测反应完毕后，加入20mL10％HCl，析出白色固体产物7a，7a干燥后溶于10mL干燥后的无水二氯甲烷，用移液管往反应瓶中加入0.1mL氯化亚砜，氮气保护，45℃冷凝回流。反应3h，TLC检测反应完后，直接用旋蒸旋干溶剂。第二步准备另一个反应瓶，加入144mg(2.05mmol)盐酸羟胺，82mg(2.05mmol)氢氧化钠，8mLTHF，0.5mLH 2O，室温搅拌。将第二步产物溶于5mL无水THF中，用恒压滴液漏斗缓慢逐滴到反应瓶中。滴加完毕后，室温搅拌3h，TLC检测反应完毕。用10％的稀HCl调节PH，调整到溶液PH到中性，析出大量白色固体，冷藏一段时间后，抽滤，得到98mg白色固体产物，收率61％。 1H NMR(500MHz,Chloroform-d)δ8.79(d,J＝4.9Hz,1H),8.18(d,J＝1.5Hz,1H),8.05(s,1H),7.82(dd,J＝7.5,1.5Hz,1H),7.78(d,J＝7.4Hz,1H),7.62–7.57(m,2H),7.42(d,J＝0.8Hz,4H),7.03–6.97(m,2H),5.05(s,2H).ESI-MS m/z：392.1[M-H] -
其他化合物的合成参考实施例1
I-2
Figure PCTCN2021079482-appb-000008
参考I-1构建苯并吡唑骨架的方法，先取代反应上癸烷链，再闭合环化，再参照I-1的合成方法得到白色固体产物460mg(I-2)，收率71％。 1H NMR((400MHz,DMSO-d 6δ11.41(s,1H),9.27(s,1H),8.48(s,1H),8.26(s,1H),7.8(m,2H),4.24(t,J＝7.1Hz,2H),1.40–1.22(m,16H),0.93–0.84(m,3H)，ESI-MS m/z：318.2[M+H] +
Figure PCTCN2021079482-appb-000009
参照I-1的合成方法，得到白色固体779mg(I-3)，收率64％。 1H NMR(400MHz,DMSO-d 6)δ11.45(s,1H),9.33(s,1H),8.22–6.28(m,12H),5.41(s,2H)，ESI-MS m/z：428.2[M+H] +
Figure PCTCN2021079482-appb-000010
参照I-1的合成方法，得到白色固体产物228mg(I-6)，收率59％。 1H NMR(400MHz,DMSO-d 6)δ11.40(s,1H),9.34(s,1H),8.12–7.01(m,12H),5.36(s,2H)，ESI-MS m/z：428.1[M+H] +
Figure PCTCN2021079482-appb-000011
参照I-1的合成方法，得到灰白色固体产物150mg(I-7)，收率56％。 1HNMR(400MHz,DMSO-d 6)δ11.29(s,1H),9.03(s,1H),8.79–6.96(m,11H),5.48(s,2H)，ESI-MS m/z：462.0[M+H] +
Figure PCTCN2021079482-appb-000012
参照I-1的合成方法，得到灰白色固体产物144mg，收率57％。 1H NMR(400MHz,DMSO-d 6)δ11.36(s,1H),9.15(s,1H),8.82-7.06(m,11H),5.44(s,2H)，ESI-MS m/z：494.0[M-H] -。
Figure PCTCN2021079482-appb-000013
参考I系列合成苯并吡唑骨架的方法，先与4-氨基-1-丁醇取代后还原为3e，再成环，与4-三氟甲基苄溴取代生成4e，参照I-1的合成方法，得到红色固体产物112mg(I-4)，收率47％。 1H NMR(400MHz,DMSO-d 6)δ11.27(s,1H),9.17(s,1H),8.26(s,1H),8.02(s,1H),7.77(d,J＝1.3Hz,2H),7.64–7.58(m,2H),7.31(m,2H),4.47(t,J＝1.0Hz,2H),4.29(t,2H),3.48(t,J＝7.1Hz,2H),1.96(m,2H),1.79(m,2H)，ESI-MS m/z：408.2[M+H] +
Figure PCTCN2021079482-appb-000014
参照I-4的合成方法，得到红色固体产物86mg(I-5)，收率41％。 1H NMR(400MHz,DMSO-d 6)δ11.29(s,1H),9.05(s,1H),8.37(s,1H),8.09(s,1H),7.70(m,2H),7.54(d,J＝7.9Hz,2H),7.45(s,1H),7.33(s,1H),7.20(s,1H),4.55(m,6H),1.23(s,2H)，ESI-MS m/z：394.1[M+H] +。
实施例2化合物抑制酸性鞘磷脂酶活性实验
酸性鞘磷脂酶可在细胞内水解鞘磷脂生成神经酰胺，针对一定量的荧光标记的反应底物(美国Avanti公司)，不同的酶活性催化生成不同量的产物，通过检测产物的含量可以考察酶活性的高低。本发明依据此原理进行实验设计。
提取已培养好的细胞中的蛋白，加入缓冲液、荧光标记的反应底物，然后分别加入不同浓度的化合物，设置空白对照组，反应结束后进行荧光分析，最后计算化合物的IC50值。
本发明部分化合物酶活性测试结果
化合物 IC50(μM) 化合物 IC50(μM)
I-1 0.47 I-5 2.78
I-2 6.58 I-6 1.24
I-3 3.68 I-7 0.39
I-4 1.34 I-8 1.16
实施例3选取活性化合物I-1进行抗抑郁活性测试
本发明通过利血平建立大鼠慢性抑郁模型(参照文献：NeurotoxRes，2014，doi：10.1007/s12640-013-9454-8)。具体实验步骤如下：
正常组随机选取8只大鼠，正常饲养。其余各组大鼠腹腔注射利血平0.2mg/Kg，每天一次持续三天。其余各组腹腔注射给予大鼠不同剂量的化合物I-1(3mg/Kg,6mg/Kg,12mg/Kg)或者阳性药阿米替林(6mg/Kg)/或不给予任何药物。给药两周后，依次进行敞箱实验(参照文献：中国药房,2016,27(19):2697-2699.)，实验结束之后进行糖水偏好实验。
如图1中A-D所示，敞箱结果显示，给予化合物I-1后，能明显改善大鼠在中央格停留时间、方格穿行次数、直立次数和梳理次数。使用高剂量(12mg/kg)I-1大鼠在敞箱中的行为指标与阳性对照组阿米替林(6mg/kg)效果相当。以上数据表明，化合物I-1具有显著的抗抑郁活性。
旷场实验结束后，所有大鼠首先训练饮用10g/L蔗糖水，即在开始的48h内用10g/L蔗糖水代替自来水，接着禁水禁食20h，然后给予10g/L蔗糖水饮用并计算24h的饮用量。
见图2，糖水消耗实验结果表明，给予大鼠I-1后，能明显恢复大鼠对糖水的摄取能力，其中高剂量组(12mg/kg)I-1略低于阳性对照药阿米替林(6mg/kg)，但是与模型组相比较有显著性差异(p＜0.01)，说明I-1在糖水消耗实验中表现出一定的抗抑郁活性。
实施例4化合物(I-1)抗动脉粥样硬化药效
Apo E是血浆脂蛋白中重要的成分,在调节血浆胆固醇水平中起重要的作用,是高脂血症、动脉粥样硬化等发生、发展的一个重要的分子靶标，ApoE基因敲除小鼠的AS病变的发展同人类极其相似.目前，ApoE基因敲除小鼠已成为研究AS发病机制以及抗AS药理学研究等方面最重要的动物模型之一。
给予APOE基因敲除小鼠高脂饮食8周，建立动脉粥样硬化模型(高脂膳食喂养的小鼠是研究动脉粥样硬化的常用动物模型，造模方法参见(Chinese Journal of Integrative Medicine,2019,25(02):108-115.)。
(1)给予西方饮食(21％脂肪+0.15％胆固醇)喂养12周后,其血脂水平显著增高,在主动脉根部位置,管壁有增厚,斑块也有明显增多,表现出典型的动脉粥样硬化病理特征。
(2)给予ApoE-/-小鼠高脂饲料(含18％氢化可可脂、0.15％胆固醇、7％酪蛋白、7％蔗糖和3％麦芽糊精)喂养8周后,通过对小鼠心脏主动脉瓣行苏木精-伊红(HE)染色,证实动脉粥样硬化模型制备成功。(检测方法参见Biomedicine&Pharmacotherapy,2018,97.)
(3)给予小鼠高脂喂养2周,行右侧颈动脉套管术后8周,发现其TC、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)等明显升高,颈总动脉套管处有明显的动脉粥样硬化斑块形成。(解剖方法参见解剖学杂志,2018,41(01):16-19)。将小鼠随机分为6组，每组5只鼠。采用腹腔注射给药方式，各组分别给予空白溶剂、空白溶剂、12mg/Kg的化合物I-1、40mg/Kg的化合物I-1。每天给药一次，共给药8周。实验结束后处死动物，分离大动脉。
将大动脉血管直接显微镜观察并拍照。结果如图3和图4所示，未给药组主动脉弓处具有明显的的斑块出现，而给予不同剂量的I-1后，斑块面积显著降低。
将分离出的动脉血管用油红O进行脂质染色，显微镜拍照后进行图像分析。结果如 图5和图6所示，化合物I-1在12mg/Kg和40mg/Kg均可以显著降低动脉粥样硬化的斑块面积，但是整体没有呈计量依赖。
分析血浆中血脂指标，结果如图7所示，与未给药的Control组相比，发现I-1对血浆总胆固醇、总甘油三酯和高密度脂蛋白(HDL)无影响，但是显著升高了低密度脂蛋白(LDL)的含量。

Claims (5)

  1. 一种含有异羟肟酸的化合物，其结构由式I所示:
    Figure PCTCN2021079482-appb-100001
    或式(I)化合物的药学上可接受的盐或前药；其中，式I中：
    R 1为C 1-C 10的饱和烷基直链、C 1-C 10的不饱和不饱和直链烷基、C 3-C 10饱和支链烷基、C 3-C 10不饱和支链烷基、C 3-C 10环烷基或苯基；
    X为C、N、O或S；
    C nH m为C 1-C 4的饱和烷基直链、C 1-C 4的不饱和直链烷基、C 1-C 4的饱和支链烷基、C 1-C 4的不饱和支链烷基，其中，1≤n≤4，2≤m≤8；
    R 2是单取代或双取代的苯基，其中取代基分别位于苯环的邻位、间位或对位，取代基为氢、氟、氯、溴、氰基、OR 3、CF 3、SF 5；R 2也可为吡啶-2-基、吡啶-3-基、吡啶-4-基、C 1-C 10的饱和烷基直链、C 1-C 10的不饱和直链烷基、C 3-C 10饱和支链烷基或C 3-C 10不饱和支链烷基；其中OR 3中的R 3为H、C 1-C 4的直链或支链烷基、C 3-C 6的环烷基、苯基；
    W、Y或Z为C、N、O和S中的任意一种元素；
    R 4、R 5或R 6为H、F、Cl、Br、CF 3、CHF 2、COCF 3、COCH 3,COC 2H 5,CN；C 1-C 6烷氧基、C1-C6的饱和烷基直链、C 1-C 6的不饱和直链烷基、C 3-C 6饱和支链烷基、C 3-C 6不饱和支链烷基及C3-C6环烷烃基中任意一种。
  2. 根据权利要求1所述的化合物，其特征在于R 1为苯基、丙基或正丁基；R 2选为4-氯苯基、4-三氟甲基苯基、3-三氟甲基苯基、4-氯-3-三氟甲基-苯基或2，4-双(三氟甲基)-苯基；X为C或O；W或Z为N；Y为C，R 4、R 5、R 6选为H。
  3. 根据权利要求1或2所述的化合物，其特征在于所述化合物如下结构任意一种：
    Figure PCTCN2021079482-appb-100002
  4. 根据权利要求1-3所述化合物在制备酸性鞘磷脂酶抑制剂中的应用。
  5. 根据权利要求1-3所述化合物在制备用于治疗动脉粥样硬化、糖尿病、肺气肿、肺水肿、肺部纤维化、慢阻肺、肺动脉高压、囊性纤维化、非酒精性脂肪肝、阿尔兹海默、多发性硬化症、脑卒中或抑郁症药物中的应用。
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