WO2022156404A1 - 一种苯并咪唑类化合物及其制备方法和在制备铁死亡抑制剂中的应用 - Google Patents

一种苯并咪唑类化合物及其制备方法和在制备铁死亡抑制剂中的应用 Download PDF

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WO2022156404A1
WO2022156404A1 PCT/CN2021/136185 CN2021136185W WO2022156404A1 WO 2022156404 A1 WO2022156404 A1 WO 2022156404A1 CN 2021136185 W CN2021136185 W CN 2021136185W WO 2022156404 A1 WO2022156404 A1 WO 2022156404A1
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ferroptosis
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顾琼
徐峻
房玉影
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Sun Yat Sen University
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    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the field of medicinal chemistry, in particular to a benzimidazole compound, a preparation method thereof, and an application in the preparation of a ferroptosis inhibitor.
  • Ferroptosis is a new type of non-apoptotic and regulated cell death discovered in recent years, characterized by iron-dependent accumulation of lipid ROS.
  • Glutathione peroxidase 4 GPX4
  • GPX4 Glutathione peroxidase 4
  • GPX4 is a key ferroptosis inhibitory protein in the ferroptosis pathway, which uses reduced glutathione as a cofactor to convert toxic lipid hydroperoxides. Reduced to non-toxic lipid alcohols. But when GPX4 is inactivated, intracellular lipid hydroperoxides accumulate in large quantities, thereby inducing ferroptosis.
  • Cysteine is a key raw material for the synthesis of reduced glutathione, and the source of intracellular cysteine depends on the glutamate/cystine antiporter system x c - .
  • the extracellular cystine is taken up by cells into the cell under the mediation of system x c - and then reduced to cysteine.
  • Inhibition of system x c - causes cysteine depletion, inhibits glutathione synthesis, and thus indirectly inhibits GPX4 enzymatic activity, ultimately inducing ferroptosis. Therefore, direct inhibition of GPX4 enzymatic activity, GPX4 knockdown, or inhibition of system x c- can induce ferroptosis.
  • ferroptosis is closely related to the occurrence and development of various neurological diseases (such as neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, Huntington's disease, etc.) and stroke, etc.)
  • Ferroptosis is expected to be an effective strategy to prevent or treat these diseases; and ferroptosis inhibitors have been reported to significantly inhibit Parkinson's disease, Huntington's disease, ischemic stroke and hemorrhagic stroke in pathological models such as Cell damage and death.
  • ferroptosis inhibitors only radical-trapping antioxidants (RTAs), lipoxygenase inhibitors, and acyl-CoA synthase long-chain family members 4 (acyl- CoA synthetase long chain family member 4, ACSL4) inhibitors, iron chelators and deuterated polyunsaturated fatty acids. Therefore, the development of novel ferroptosis inhibitors is urgently needed.
  • the object of the present invention is to overcome the defects or deficiencies that the existing ferroptosis inhibitors have less types and poor activity, and provide a benzimidazole compound.
  • the benzimidazole compounds provided by the present invention have good ferroptosis inhibitory activity by introducing a lipophilic group at the R 1 position and a specific group at the R 2 position, and can be used for preventing and treating nervous system diseases such as A lead compound in stroke.
  • Another object of the present invention is to provide a method for preparing the above-mentioned benzimidazole compounds.
  • Another object of the present invention is to provide the application of the above-mentioned benzimidazole compounds in the preparation of ferroptosis inhibitors.
  • the present invention provides the following technical solutions:
  • R is halogen, methyl, alkoxy, alkynyloxy or hydroxy
  • X is C or N.
  • the present invention synthesized a series of benzimidazole compounds (formula (I)) by introducing a lipophilic group at the R1 position.
  • the lipophilic group at the R 1 position includes the following four different cyclic aliphatic amine side chains: N-methylpiperazine, methylpiperidine, piperidine and morpholine, and the middle aromatic ring is a benzene ring or pyridine ;
  • Determination of the inhibitory activity of this series of benzimidazole compounds on ferroptosis shows that R 1 is When X is C, the inhibitory activity is the best; therefore, based on this, different groups are introduced at the R 2 position to obtain a series of benzimidazole compounds (such as formula (II)).
  • the benzimidazole compounds provided by the invention have good ferroptosis inhibitory activity, and can be used as a leading compound for preventing and treating nervous system diseases such as stroke.
  • the halogen is F or Cl.
  • the alkoxy group is -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 CH 3 or -OCH(CH 3 ) 2 .
  • the alkynyloxy group is -OCH 2 C ⁇ CH.
  • R is substituted at one or both of the 6, 7 or 8 positions.
  • R when R is substituted at the 6-position, R is -F, -Cl, -OCH 3 , -OH, -OCH 2 CH 3 , -OCH 2 CH 2 CH 3 , -OCH(CH 3 ) 2 or - OCH 2 C ⁇ CH;
  • R is -CH 3 when R is substituted at the 7 position
  • R is -OCH 3 or -OH
  • R is substituted at the 6- and 7-positions, R is -OH or -OCH 3 .
  • R is -OH or -OCH 3 .
  • the present invention also provides a method for preparing the above-mentioned benzimidazole compounds.
  • the preparation method of above-mentioned benzimidazole compound comprises the steps:
  • the conditions of the coupling reaction in S11 are: basic conditions, and the reaction is carried out at 110° C. for 16 hours.
  • the reduction temperature in S12 is 60° C. and the time is 2 h.
  • the amide reaction in S13 is stirring at room temperature for 6 hours, and the subsequent ring-closing reaction is refluxing overnight under glacial acetic acid.
  • a mixture of dichloromethane and methanesulfonic acid is used as a solvent.
  • lithium aluminum hydride is used for reduction in S22.
  • the reaction is carried out at 80° C. for 24 hours.
  • the conditions of the amide reaction and the ring closure reaction in S25 are the same as those in S13.
  • the conditions of the coupling reaction in S31 are as follows: in an inert atmosphere, the reaction is carried out at 80° C. for 24 h or 16 h.
  • the conditions of the amide reaction and the ring closure reaction in S33 are the same as those in S13.
  • the use of the benzimidazole compounds in the preparation of ischemic stroke medicines is preferred.
  • the compounds provided by the present invention are tested for the activity of inhibiting ferroptosis by using Erastin to induce ferroptosis on HT22 cells, and it is found that the activity of compound 9a is the best.
  • 9a has significant inhibitory activity against ferroptosis induced by different types of ferroptosis inducers, and has inhibitory effects on two hallmark features of ferroptosis (lipid peroxidation and up-regulation of PTGS2 mRNA).
  • the present invention has the following advantages and effects:
  • the benzimidazole compounds provided by the invention have significant ferroptosis inhibitory activity, and the benzimidazole ferroptosis inhibitors have great application prospects in the prevention and treatment of nervous system diseases such as stroke;
  • the preparation method of the present invention has easily available raw materials and simple preparation.
  • Figure 1 shows the inhibitory effect of compound 9a on two features of ferroptosis (RSL3-induced lipid peroxidation and PTGS2 mRNA up-regulation);
  • FIG. 1 shows the TTC detection results.
  • the present invention is further explained below with reference to the embodiments and the accompanying drawings, but the embodiments do not limit the present invention in any form.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • reaction formula 1 (a) potassium carbonate, N,N-dimethylformamide, 110°C, 16h; (b) nickel, hydrazine hydrate, methanol, 60°C, 2h; (c) 1.O-benzo Triazole-N,N,N',N'-tetramethylurea tetrafluoroborate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 6h; 2 . Glacial acetic acid, reflux, overnight.
  • Triazole-N,N,N',N'-tetramethylurea tetrafluoroborate (385mg, 1.2mmol)
  • o-phenylenediamine derivatives 2a-2d (1.1mmol) were added , at room temperature for 6 hours.
  • the reaction was quenched with ice water, extracted with ethyl acetate, the organic layer was dried over anhydrous sodium sulfate, and spin-dried under reduced pressure; the solid obtained by spin-drying was dissolved in 10 mL of glacial acetic acid, and the reaction was refluxed overnight.
  • intermediates 7a-7i are hydrolyzed under alkaline conditions to obtain the intermediates 8a-8i, which are then reacted with the intermediate o-phenylenediamine derivative 2a through amide reaction and ring closure reaction, and finally the final products 9a-9k are obtained.
  • intermediates 5a-5b, 6a-6b, 7a and 7c were synthesized according to the conditions in our previous work.
  • reaction formula 2 (a) methanesulfonic acid, sodium azide, dichloromethane, room temperature, overnight; (b) lithium aluminum hydride, tetrahydrofuran, reflux, 4h; (c) potassium carbonate, cuprous iodide, L-proline, dimethyl sulfoxide, 80°C, 24h, nitrogen protection; (d) lithium hydroxide, tetrahydrofuran, 90°C, 3h; (e) 1.O-benzotriazole-N,N ,N',N'-tetramethylurea tetrafluoroborate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 6h; 2. Glacial acetic acid, reflux, overnight . Compounds 9j and 9k were obtained by demethylation of compounds 9h and 9i under boron tribromide, respectively.
  • intermediate 7b was synthesized from 6-chloro-1,2,3,4-tetrahydroisoquinoline (5.03g, 30mmol) and ethyl 3-iodobenzoate (5.52g, 20mmol) according to the above method to obtain a white oil material (3.72 g, 59%).
  • intermediate 7d was synthesized from 1,2,3,4-tetrahydroisoquinolin-6-ol (4.48g, 30mmol) and ethyl 3-iodobenzoate (5.52g, 20mmol) according to the above method to obtain a white oil material (3.33 g, 56%).
  • intermediate 7g was synthesized from 1,2,3,4-tetrahydroisoquinolin-8-ol (4.48g, 30mmol) and ethyl 3-iodobenzoate (5.52g, 20mmol) according to the above method to obtain a white oil material (3.98 g, 67%).
  • reaction formula 3 (a) di-tert-butyl dicarbonate, 4-dimethylaminopyridine, tetrahydrofuran, room temperature, 0.5h; (b) sodium hydride, acetonitrile, 60°C, 24h; (c) 1. hydrochloric acid, 1 ,4-dioxane, room temperature, 3h; 2.
  • Potassium carbonate cuprous iodide, L-proline, dimethyl sulfoxide, 80°C, 24h, nitrogen protection;
  • reaction formula 4 (a) potassium carbonate, cuprous iodide, L-proline, dimethyl sulfoxide, 80°C, 24h; or sodium carbonate, tetrakistriphenylphosphonium palladium, 1,4-dioxane Hexacyclic/water, 80°C, 16h, nitrogen protection; (b) lithium hydroxide, tetrahydrofuran, 90°C, 3h; (c) 1.O-benzotriazole-N,N,N',N'- Tetramethylurea tetrafluoroborate, N,N-diisopropylethylamine, N,N-dimethylformamide, room temperature, 6h; 2. Glacial acetic acid, reflux, overnight.
  • 17b was synthesized from 16b and 2a as above as a white solid (40%).
  • 1 H NMR 400MHz, CD 3 OD
  • 17c was synthesized from 16c and 2a as above as a tan solid (44%).
  • 1 H NMR 400MHz, CD 3 OD
  • ⁇ 8.24 s, 1H
  • 7.54-7.49 m, 2H
  • 7.19 s, 1H
  • 7.18-7.16 m, 1H
  • 7.04(dd, J 8.8, 2.0Hz, 1H)
  • Example 2 Test of ferroptosis inhibitory activity of the compounds obtained in Example 1
  • HT22 cells mouse hippocampal neuron cells
  • DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody at 37°C and 5% carbon dioxide concentration.
  • HT22 cells were seeded in a 96-well plate at 3000 cells/well, and after culturing for 24 hours, the original culture medium was removed by suction, 1 ⁇ M RSL3 ⁇ compound was added for 24 hours, and 10 ⁇ l CCK-8 solution was added to each well, and incubated at 37°C for 4 hours. , and the absorbance OD value was detected at a wavelength of 450 nm.
  • HT1080 cells human fibrosarcoma cells
  • MEM medium containing 10% fetal bovine serum and 1% double antibody at 37°C and 5% carbon dioxide concentration.
  • 50nM siRNA scramble and siRNA GPX4 were transferred into HT1080 cells with Lipofectamine TM 3000 reagent, respectively. Compounds were added after 12 hours, and the cells were harvested after incubation for 36 hours for qPCR and CCK-8 experiments.
  • the specific steps of the qPCR experiment are as follows: the cells to be treated were washed twice with PBS, the total RNA was extracted with RNAiso plus reagent, 1 ⁇ g of total RNA was reverse transcribed into cDNA using ReverTra Ace qPCR RT Master Mix, and then PCR was performed using SYBR Green Realtime PCR Master Mix. Amplification.
  • HT22 cells were seeded in 6-well plates at 3 ⁇ 10 4 cells/well for 24 hours, then 1 ⁇ M RSL3 ⁇ compound was added for 3 hours, and then incubated with 5 ⁇ M BODIPY-C11 probe at 37°C for half an hour. After cells were washed once with PBS, trypsinized, centrifuged and collected in 1 ml of PBS, the fluorescence intensity of the FL1 channel was detected by flow cytometry at 488 nm. At least 10,000 cells were detected per sample.
  • HT22 cells were seeded at 3000 cells/well in a 96-well plate and cultured for 24 hours, then 1 ⁇ M RSL3 ⁇ compound was added for 3 hours, and then 5 ⁇ M BODIPY-C11 probe and Hoechst 33342 (1 ⁇ g/ml) were used at 37°C After co-incubating for half an hour, the cells were washed once with PBS, and then photographed with a FV3000 laser confocal microscope (60 ⁇ ).
  • HT22 cells were seeded at 3 ⁇ 10 4 cells/well in 6-well plates and cultured for 24 hours, then added 1 ⁇ M RSL3 ⁇ compound for 12 hours, washed twice with PBS, extracted total RNA with RNAiso plus reagent, and used ReverTra Ace qPCR RT Master Mix 1 ⁇ g of total RNA was reverse transcribed into cDNA, and then used SYBR Green Realtime PCR Master Mix for PCR amplification.
  • compound 9a inhibited RSL3-induced ferroptosis in HT22 cells in a dose-dependent manner; compound 9a also inhibited the ferroptosis induced by GPX4 in HT1080 cells in a dose-dependent manner.
  • Activity live cell imaging results showed that compound 9a could significantly inhibit the increase of RSL3-induced lipid ROS, which was consistent with the results of flow cytometry.
  • compound 9a also had inhibitory activity on the increase of RSL3-induced cytoplasmic ROS; finally, compound 9a also had inhibitory activity. Concentration-dependently inhibited RSL3-induced upregulation of PTGS2 mRNA.
  • Example 4 Relief effect of compound 9a obtained in Example 1 on ischemia-reperfusion injury in vivo
  • Ischemic stroke is the most common form of stroke and one of the leading causes of death and permanent disability. So far, there is no effective treatment.
  • ferroptosis is involved in neuronal damage and death in the pathological process of ischemic stroke, and inhibition of ferroptosis can alleviate brain damage in vivo.
  • compound 9a has significant ferroptosis inhibitory activity on HT22 cells, we investigated whether this compound can protect SD rats from ischemia/reperfusion in the Middle Cerebral Artery Occlusion (MCAO) model nerve damage caused.
  • MCAO Middle Cerebral Artery Occlusion
  • the animals were randomly divided into 4 groups, namely blank control group, model control group, positive control group, and test sample 9a group, with 12 animals in each group.
  • Dosage test sample 9a 10 mg/kg, positive control drug edaravone 10 mg/kg; blank control group and model control group were given normal saline in the same way.
  • Dosing frequency once half an hour before the operation, once 2 hours after the operation; administration route: intraperitoneal injection, the volume is 5ml/kg.
  • the rats were anesthetized by intraperitoneal injection of 7% chloral hydrate 5ml/kg, the median neck incision was made, the left common carotid artery and external carotid artery were isolated and ligated, and the pterygofrontal artery was isolated.
  • the proximal end of the internal carotid artery is prepared, and the distal end is placed with an arterial clip.
  • An incision is made at the bifurcation of the common carotid artery, and a 4-0 nylon thread is inserted with a depth of 17 to 20 mm. , block all blood flow sources in the middle cerebral artery, let it be ischemia for 1.5h and then pull out the suture for about 1cm to achieve reperfusion.
  • the wound was disinfected with iodophor, and the skin was sutured. Then return to the cage.
  • the rest of the steps were the same as above, except that the line was not plugged in.
  • the results of TTC staining showed that compared with the model group, the cerebral infarct volume was significantly reduced and the behavior was more normal after 9a treatment, indicating that compound 9a can significantly alleviate ischemia/reperfusion-induced brain injury in vivo, and is expected to develop into a Lead compounds for the treatment of ischemic stroke.

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Abstract

本发明公开了一种苯并咪唑类化合物及其制备方法和在制备铁死亡抑制剂中的应用。该苯并咪唑类化合物具有如式(Ⅰ)或式(Ⅱ)所示结构：本发明提供的苯并咪唑类化合物通过在R 1位置引入亲脂性基团，及在R 2位置引入特定的基团，得到的化合物具有较好的铁死亡抑制活性，可作为防治神经系统疾病如中风的一种先导化合物。

Description

一种苯并咪唑类化合物及其制备方法和在制备铁死亡抑制剂中的应用 技术领域
本发明涉及药物化学领域，特别涉及一种苯并咪唑类化合物及其制备方法和在制备铁死亡抑制剂中的应用。
背景技术
铁死亡(ferroptosis)是近年来发现的一种非凋亡的、可调控的新型细胞死亡类型，以依赖于铁的脂质ROS堆积为特征。谷胱甘肽过氧化物酶4(Glutathione peroxidase 4,GPX4)是铁死亡通路中一个关键的铁死亡抑制蛋白，其利用还原型谷胱甘肽作为辅因子，将有毒的脂质氢过氧化物还原为无毒的脂质醇。但当GPX4失活时，细胞内脂质氢过氧化物大量堆积，从而诱导铁死亡。半胱氨酸是合成还原型谷胱甘肽的一个关键原料，而细胞内半胱氨酸的来源依赖于谷氨酸/胱氨酸反向转运子system x c -。胞外的胱氨酸在system x c -介导下，被细胞摄取进入到胞内，然后被还原为半胱氨酸。抑制system x c -会造成半胱氨酸耗竭，抑制谷胱甘肽的合成，从而间接抑制GPX4酶活，最终诱导铁死亡。因此，直接抑制GPX4酶活、敲除GPX4或者抑制system x c -都可以诱导铁死亡。
越来越多的研究表明，铁死亡与多种神经系统疾病(如神经退行性疾病(帕金森症、阿尔茨海默症、亨廷顿舞蹈症等)和中风等)的发生和发展密切相关，抑制铁死亡有望成为预防或者治疗这些疾病的一种有效策略；并且铁死亡抑制剂在帕金森症、亨廷顿舞蹈症、缺血性脑中风和出血性脑中风等病理模型中，均被报道能够显著抑制细胞损伤和死亡。但目前已有的铁死亡抑制剂种类较少，仅有自由基清除抗氧化剂(radical-trapping antioxidants,RTAs)、脂氧合酶抑制剂、酯酰辅酶A合成酶长链家族成员4(acyl-CoA synthetase long chain family member 4,ACSL4)抑制剂、铁螯合剂和氘代多不饱和脂肪酸这五大类。因此，急需发展新型的铁死亡抑制剂。
已有研究(Y.Fang,H.Zhou,Q.Gu,J.Xu,Synthesis and evaluation of tetrahydroisoquinoline-benzimidazole hybrids as multifunctional agents for the treatment of Alzheimer's disease,European journal of medicinal chemistry,167(2019)133-145，本发明人前期研究工作)发现在小鼠海马神经元HT22细胞上，10个苯并咪唑衍生物能够抑制高浓度谷氨酸诱导的铁死亡，随后我们发现这些化合物也可以阻断system x c -抑制剂Erastin诱导的铁死亡，但该类苯并咪唑衍生物对铁死亡的抑制活性不佳。
因此，开发新的具有较好抑制活性的铁死亡抑制剂具有重要的研究意义和应用价值。
发明内容
本发明的目的在于克服现有铁死亡抑制剂种类较少，活性不佳的缺陷或不足，提供一种苯并咪唑类化合物。本发明提供的苯并咪唑类化合物通过在R 1位置引入亲脂性基团，及在R 2位置引入特定的基团，得到的化合物具有较好的铁死亡抑制活性，可作为防治神经系统疾病如中风的一种先导化合物。
本发明的另一目的在于提供上述苯并咪唑类化合物的制备方法。
本发明的另一目的在于提供上述苯并咪唑类化合物在制备铁死亡抑制剂中的应用。
为了实现本发明的上述目的，本发明提供了如下技术方案：
一种苯并咪唑类化合物，具有如式(Ⅰ)或式(Ⅱ)所示结构：
Figure PCTCN2021136185-appb-000001
其中，R 1为
Figure PCTCN2021136185-appb-000002
R 2为
Figure PCTCN2021136185-appb-000003
R为卤素、甲基、烷氧基、炔氧基或羟基；
X为C或N。
我们前期工作中发现在小鼠海马神经元HT22细胞上，10个苯并咪唑衍生物能够抑制高浓度谷氨酸诱导的铁死亡，随后我们发现这些化合物也可以阻断system x c -抑制剂Erastin诱导的铁死亡。但该类苯并咪唑衍生物对铁死亡的抑制活性不佳，于是本发明通过结构改造，得到具有更为优异的铁死亡抑制活性的苯并咪唑类化合物，可将其运用于神经系统疾病的预防或者治疗中。
具体地，本发明通过在R 1位置引入亲脂性基团，合成了一系列苯并咪唑类化合物(式(Ⅰ))。其中R 1位置的亲脂性基团包括以下四种不同的环状脂肪胺基侧链：N-甲基哌嗪、甲基哌啶、哌啶和吗啉，中间的芳香环是苯环或吡啶；对这系列苯并咪唑类化合物对铁死亡的抑制活性进行测定表明，R 1为
Figure PCTCN2021136185-appb-000004
X为C时抑制活性最佳；故以此为基础，在R 2位置引入不同的基团，进而得到一系列苯并咪唑类化合物(如式(Ⅱ))。
本发明提供的苯并咪唑类化合物具有较好的铁死亡抑制活性，可作为防治神经系统疾病如中风的一种先导化合物。
优选地，所述卤素为F或Cl。
优选地，所述烷氧基为-OCH 3、-OCH 2CH 3、-OCH 2CH 2CH 3或-OCH(CH 3) 2。
优选地，所述炔氧基为-OCH 2C≡CH。
优选地，R在6位、7位或8位中的一位或两位为取代。
更为优选地，R在6位取代时，R为-F、-Cl、-OCH 3、-OH、-OCH 2CH 3、-OCH 2CH 2CH 3、-OCH(CH 3) 2或-OCH 2C≡CH；
R在7位取代时R为-CH 3；
R在8位取代时，R为-OCH 3或-OH；
R在6位和7位取代时，R为-OH或-OCH 3。
所述R在6位和8位取代时，R为-OH或-OCH 3。
本发明还提供上述苯并咪唑类化合物的制备方法。
当苯并咪唑类化合物具有式(Ⅰ)所示结构时，包括如下步骤：
S11：2-硝基-5-氯苯胺和环状脂肪胺R 1-H进行偶联反应，得
Figure PCTCN2021136185-appb-000005
S12：
Figure PCTCN2021136185-appb-000006
在镍和水合肼条件下还原为
Figure PCTCN2021136185-appb-000007
S13：
Figure PCTCN2021136185-appb-000008
反应，即得式(Ⅰ)所示结构的苯并咪唑类化合物
Figure PCTCN2021136185-appb-000009
上述苯并咪唑类化合物的制备方法包括如下步骤：
S11：2-硝基-5-氯苯胺和环状脂肪胺R 1-H进行偶联反应，得
Figure PCTCN2021136185-appb-000010
S12：
Figure PCTCN2021136185-appb-000011
在镍和水合肼条件下还原为
Figure PCTCN2021136185-appb-000012
S13：
Figure PCTCN2021136185-appb-000013
反应，即得式(Ⅰ)所示结构的苯并咪唑类化合物
Figure PCTCN2021136185-appb-000014
更为优选地，S11中偶联反应的条件为：碱性条件，110℃下反应16h。
更为优选地，S12中还原的温度为60℃，时间为2h。
更为优选地，S13中酰胺反应为常温搅拌6h，后续关环反应为在冰乙酸下，回流反应过夜。
当R 2为
Figure PCTCN2021136185-appb-000015
时，包括如下步骤：
S21：茚酮衍生物
Figure PCTCN2021136185-appb-000016
在酸性条件下，与叠氮化钠进行Aube-Schmidt重排反应得
Figure PCTCN2021136185-appb-000017
S22：
Figure PCTCN2021136185-appb-000018
经还原后得四氢异喹啉衍生物
Figure PCTCN2021136185-appb-000019
S23：四氢异喹啉衍生物
Figure PCTCN2021136185-appb-000020
和3-碘苯甲酸乙酯
Figure PCTCN2021136185-appb-000021
经偶联反应得
Figure PCTCN2021136185-appb-000022
S24：
Figure PCTCN2021136185-appb-000023
碱性条件下水解得
Figure PCTCN2021136185-appb-000024
S25：
Figure PCTCN2021136185-appb-000025
和邻苯二胺衍生物
Figure PCTCN2021136185-appb-000026
通过酰胺反应和关环反应即得式(Ⅱ)所示结构的苯并咪唑类化合物
Figure PCTCN2021136185-appb-000027
更为优选地，S21中以二氯甲烷和甲基磺酸的混合液作为溶剂。
更为优选地，S22中利用氢化铝锂进行还原。
更为优选地，S23中偶联反应的条件下：惰性气氛下，80℃下反应24h。
更为优选地，S25中酰胺反应和关环反应的条件同S13。
当R 2为
Figure PCTCN2021136185-appb-000028
时，包括如下步骤：
S31：3-碘苯甲酸乙酯
Figure PCTCN2021136185-appb-000029
或3-乙氧羰基苯硼酸
Figure PCTCN2021136185-appb-000030
与R 2-H或R 2-Br经偶联反应得
Figure PCTCN2021136185-appb-000031
S32：
Figure PCTCN2021136185-appb-000032
在碱性条件下水解得
Figure PCTCN2021136185-appb-000033
S33：
Figure PCTCN2021136185-appb-000034
与中间体邻苯二胺衍生物
Figure PCTCN2021136185-appb-000035
通过酰胺反应和关环反应，即得式(Ⅱ)所示结构的苯并咪唑类化合物
Figure PCTCN2021136185-appb-000036
更为优选地，S31中偶联反应的条件为：惰性气氛下，80℃下反应24h或16h。
更为优选地，S33中中酰胺反应和关环反应的条件同S13。
上述苯并咪唑类化合物在制备铁死亡抑制剂中的应用也在本发明的保护范围内。
优选地，所述苯并咪唑类化合物在制备缺血性脑中风药物中的应用。
本发明提供的化合物通过在HT22细胞上，用Erastin诱导细胞铁死亡的实验检测化合物抑制铁死亡的活性，发现化合物9a的活性最好，对其进行深入的铁死亡抑制活性的评价，研究表明化合物9a对不同类型铁死亡诱导剂诱导的铁死亡均有显著的抑制活性，并且对铁死亡的两个标志性特征(脂质过氧化和PTGS2 mRNA的上调)均有抑制效果。
相对于现有技术，本发明具有如下的优点及效果：
(1)本发明提供的苯并咪唑类化合物具有显著的铁死亡抑制活性，所述苯并咪唑类铁死亡抑制剂在预防及治疗中风等神经系统疾病中具备极大的应用前景；
(2)本发明的制备方法原料易得，制备简单。
附图说明
图1为化合物9a对铁死亡的两个特征(RSL3诱导的脂质过氧化和PTGS2 mRNA上调)的抑制效果；
图2为TTC检测结果。
具体实施方式
以下结合实施例和附图进一步解释本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
本发明实施例提供的苯并咪唑类化合物，具有化学通式A或B所示结构：
Figure PCTCN2021136185-appb-000037
实施例1化合物4a-4h(A式)、9a-9k、14a-14d和17a-17c(B式)的合成
化合物4a-4h、9a-9k、14a-14d和17a-17c的合成分别如反应式1、2、3和4所示。
按反应式1所示的合成路线进行化合物4a-4h的合成。2-硝基-5-氯苯胺与四种不同的环状脂肪胺(N-甲基哌嗪、甲基哌啶、哌啶和吗啉)通过在碱性条件下发生偶联反应，得到中间体1a-1d，接着在镍和水合肼条件下硝基被还原为氨基得到中间体2a-2d。中间体3a-3b按我们前期工作中的条件合成得到，这两个有机羧酸分别与中间体2a-2d反应得到终产物4a-4h。
Figure PCTCN2021136185-appb-000038
反应式1中：(a)碳酸钾，N,N-二甲基甲酰胺，110℃，16h；(b)镍，水合肼，甲醇，60℃，2h；(c)1.O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯，N,N-二异丙基乙胺，N,N-二甲基甲酰胺，室温，6h；2.冰乙酸，回流，过夜。
具体步骤如下：
(1)中间体1a-1d的合成：向耐压管中依次加入2-硝基-5-氯苯胺(997.6mg,5.8mmol)、环状脂肪胺(6.9mmol)和碳酸钾(1.28g,9.3mmol)，再加入干燥的N，N-二甲基甲酰胺(10mL)，油浴加热到110℃，磁力搅拌反应16小时。反应结束后，向反应体系中加入适量水，然后用乙酸乙酯萃取(3×150mL)，有机层经无水硫酸钠干燥后，减压浓缩。将旋干得到的固体经柱层析分离纯化(二氯甲烷:乙酸乙酯＝2:1)得到中间体1a-1d。
中间体1a由2-硝基-5-氯苯胺和N-甲基哌嗪按上述方法合成，得到黄色固体(0.97g,71％)。
中间体1b由2-硝基-5-氯苯胺和4-甲基哌啶按上述方法合成，得到黄色固体(1.03g,76％)。
中间体1c由2-硝基-5-氯苯胺和哌啶按上述方法合成，得到黄色固体(1.06g,83％)。
中间体1d由2-硝基-5-氯苯胺和吗啉按上述方法合成，得到黄色固体(1.02g,79％)。
(2)终产物4a-4h的合成：将中间体1a-1d(5.0mmol)溶于甲醇(30mL)中，加入水合肼(5mL)和催化量的镍，反应在60℃下磁力搅拌2小时。反应结束后，冷却至室温，过滤，将滤液浓缩得到中间体2a-2d(黑色固体，90-95％)。接着，将有机酸3a-3b(1mmol)溶于5mL N，N-二甲基甲酰胺中，冰浴下加入N，N-二异丙基乙胺(0.21mL,1.2mmol)和O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯(385mg,1.2mmol)，搅拌30分钟后，加入邻苯二胺衍生物2a-2d(1.1mmol)，室温下反应6小时。然后冰水淬灭反应，用乙酸乙酯萃取，有机层用无水硫酸钠干燥，减压旋干；将旋干得到的固体溶于10mL冰乙酸中，回流反应过夜。反应结束后，冷却至室温，用饱和的碳酸氢钠溶液调节pH至中性，乙酸乙酯萃取，有机层干燥后减压旋干，经柱层析分离纯化得到相应的终产物。
化合物4a-4h的结构、外观和核磁共振谱图数据如以下所示。
Figure PCTCN2021136185-appb-000039
4a由2a和3a按上述方法合成，白色固体(43％)。 1H NMR(400MHz,CDCl 3)δ7.74(s,1H),7.41-7.28(m,3H),7.21-7.10(m,4H),7.07-6.88(m,3H),4.47(s,2H),3.62(t,J＝5.4Hz,2H),3.23(s,4H),2.98(t,J＝5.2Hz,2H),2.65(s,4H),2.39(s,3H). 13C NMR(100MHz,DMSO-d 6)δ150.6,150.4,148.0,137.9,135.8,134.7,134.3,131.2,129.5,128.4,126.6,126.3,125.9,118.8,115.7,115.5,113.5,111.7,97.1,54.9(2C),50.2,49.7,49.7,45.8,45.6,28.2.HRMS(ESI):calcd for C 27H 29N 5[M+H] +424.2496,found 424.2521.
Figure PCTCN2021136185-appb-000040
4b由2b和3a按上述方法合成，白色固体(50％)。 1H NMR(500MHz,DMSO-d 6)δ7.72(s,1H),7.53-7.43(m,2H),7.35(t,J＝7.3Hz,1H),7.27-7.16(m,4H),7.06(d,J＝5.8Hz,1H),6.99-6.85(m,2H),4.47(s,2H),3.66-3.52(m,4H),2.96(s,2H),2.69-2.56(m,2H),1.75-1.66(m,2H),1.52-1.42(m,1H),1.35-1.24(m,2H),0.94(d,J＝6.3Hz,3H). 13C NMR(125MHz,DMSO-d 6)δ150.4,148.5,143.7,139.1,137.8,134.7,134.4,131.1,129.6,128.4,126.6,126.3,126.0,123.4,123.1,115.7,115.6,111.8,99.6,50.9,49.7(2C),45.6,34.0(2C),30.2,28.2,21.9.HRMS(ESI):calcd for C 28H 30N 4[M+H] +423.2543,found 423.2548.
Figure PCTCN2021136185-appb-000041
4c由2c和3a按上述方法合成，白色固体(48％)。 1H NMR(500MHz,DMSO-d 6)δ7.60(s,1H),7.35(d,J＝6.2Hz,2H),7.23(t,J＝6.4Hz,1H),7.17-6.92(m,6H),6.80(s,1H),4.36(s,2H),3.50(s,6H),2.84(s,2H), 1.59-1.51(m,4H),1.44-1.40(m,2H). 13C NMR(125MHz,DMSO-d 6)δ150.6,150.5,149.1,137.8,135.8,134.8,134.4,131.2,129.7,128.5,126.7,126.4,126.1,118.9,115.9,115.6,114.5,111.8,97.7,52.1,51.6,49.8,45.7,28.3,25.9,25.7,24.0.HRMS(ESI):calcd for C 27H 28N 4[M+H] +409.2387,found 409.2390.
Figure PCTCN2021136185-appb-000042
4d由2d和3a按上述方法合成，白色固体(43％)。 1H NMR(400MHz,DMSO-d 6)δ7.71(s,1H),7.48(t,J＝8.1Hz,1H),7.34(t,J＝8.0Hz,1H),7.27-7.23(m,1H),7.21-7.11(m,5H),6.99-6.88(m,2H),4.47(s,2H),3.79-3.71(m,4H),3.61(t,J＝5.8Hz,2H),3.12-3.04(m,4H),2.95(t,J＝5.7Hz,2H). 13C NMR(100MHz,DMSO-d 6)δ150.9,150.1,147.5,138.7,134.4,134.1,131.7,131.0,129.3,129.1,128.7,127.9,127.0,126.1,125.9,125.5,115.6,115.2,111.8,66.0(2C),50.1(2C),49.5,45.3,27.9.HRMS(ESI):calcd for C 26H 26N 4O[M+H] +411.2179,found 411.2196.
Figure PCTCN2021136185-appb-000043
4e由2a和3b按上述方法合成，白色固体(45％)。 1H NMR(400MHz,CD 3OD)δ7.63-7.56(m,2H),7.36-7.31(m,2H),7.26-7.21(m,4H),7.13(d,J＝8.9Hz,1H),6.93(d,J＝8.5Hz,1H),4.88(s,2H),4.03(t,J＝5.2Hz,2H),3.44-3.37(m,4H),3.09-3.02(m,6H),2.70(s,3H). 13C NMR(100MHz,CD 3OD)δ159.7,153.0,149.2,147.1,139.5,136.6,135.7,129.4,127.6,127.4,127.2,125.3,125.2,118.7,117.0,112.4,110.8,108.8,55.6(2C),50.8(2C),48.0,45.0,43.6,29.9.HRMS(ESI):calcd for C 26H 28N 6[M+H] +425.2448,found 425.2450.
Figure PCTCN2021136185-appb-000044
4f由2b和3b按上述方法合成，白色固体(50％)。 1H NMR(400MHz,CDCl 3)δ7.75(d,J＝7.3Hz,1H),7.60-7.52(m,2H),7.24-7.13(m,5H),7.05(d,J＝8.3Hz,1H),6.69(d,J＝8.5Hz,1H),4.76(s,2H),3.89(t,J＝5.8Hz,2H),3.60(d,J＝12.0Hz,2H),2.95(t,J＝5.7Hz,2H),2.73(t,J＝11.2Hz,2H),1.75(d,J＝11.2Hz,2H),1.55-1.37(m,3H),0.98(d,J＝5.8Hz,3H). 13C NMR(100MHz,CDCl 3)δ158.0,150.0,149.3,144.4,138.5,135.3,134.2,128.4,126.7,126.5,126.3,116.7,116.4,116.3,110.8,108.2,101.1,101.1,51.8,47.2,42.6,34.1(2C),30.6,29.8,29.0,21.9.HRMS(ESI):calcd for C 27H 29N 5[M+H] +424.2496,found 424.2516.
Figure PCTCN2021136185-appb-000045
4g由2c和3b按上述方法合成，白色固体(48％)。 1H NMR(500MHz,CD 3OD)δ7.64(t,1H),7.54-7.47(m,2H),7.28-7.10(m,5H),7.09-7.02(m,1H),6.86-6.80(m,1H),4.80(s,2H),3.95(t,J＝5.8Hz,2H),3.11(s,4H),2.96(t,J＝5.5Hz,2H),1.80-1.69(m,4H),1.62-1.53(m,2H). 13C NMR(100MHz,DMSO-d 6)δ157.8,150.0,149.1,146.6,146.6,138.4,138.0,135.5,135.1,134.5,128.4,126.5,126.2,125.9,119.2,114.7,109.2,106.9,97.8,51.3(2C),46.6,41.9,28.1,25.6(2C),23.9.HRMS(ESI):calcd for C 26H 27N 5[M+H] +410.2339,found 410.2349.
Figure PCTCN2021136185-appb-000046
4h由2d和3b按上述方法合成，白色固体(56％)。 1H NMR(400MHz,CDCl 3)δ7.74-7.61(m,3H),7.25-7.18(m,4H),7.01(d,J＝7.9Hz,2H),6.75(d,J＝8.4Hz,1H),4.79(s,2H),3.95(t,J＝5.9Hz,2H),3.91(t,J＝4.6Hz,4H),3.19(t,J＝4.4Hz,4H),3.02(t,J＝5.8Hz,2H). 13C NMR(100MHz,DMSO-d 6)δ157.9,150.3,148.3,146.6,138.5,138.4,135.5,135.1,134.6,128.5,126.6,126.3,126.0,119.3,113.5,109.3,107.1,97.3,66.3(2C),50.5,50.0(2C),46.5,28.1.HRMS(ESI):calcd for C 25H 25N 5O[M+H] +412.2132,found 412.2151.
按反应式2所示的合成路线进行化合物9a-9k的合成。茚酮衍生物在酸性条件下，与叠氮化钠进行Aube-Schmidt重排反应得到中间体5a-5d，然后该中间体被氢化铝锂还原，得到四氢异喹啉衍生物6a-6d。此合成的以及可购买得到的四氢异喹啉衍生物与3-碘苯甲酸乙酯经偶联反应，得到中间体7a-7i。接着中间体7a-7i在碱性条件下水解得到中间体8a-8i，接着与中间体邻苯二胺衍生物2a通过酰胺反应和关环反应，最终得到终产物9a-9k。其中，中间体5a-5b、6a-6b、7a和7c按我们前期工作中的条件合成得到。
Figure PCTCN2021136185-appb-000047
反应式2中：(a)甲基磺酸，叠氮化钠，二氯甲烷，室温，过夜；(b)氢化铝锂，四氢呋喃，回流，4h；(c)碳酸钾，碘化亚铜，L-脯氨酸，二甲基亚砜，80℃，24h，氮气保护；(d)氢氧化锂，四氢呋喃，90℃， 3h；(e)1.O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯，N，N-二异丙基乙胺，N,N-二甲基甲酰胺，室温，6h；2.冰乙酸，回流，过夜。其中化合物9j和9k分别由化合物9h和9i在三溴化硼条件下脱甲基得到。
具体步骤如下：
(1)中间体5a-5d的合成：原料7-甲氧基-1-茚酮(4.31g,26.6mmol)溶解在40mL二氯甲烷和40mL甲基磺酸的混合液中，冷却到0℃后，将叠氮化钠(3.46g,53.2mmol)缓慢加入反应瓶中，室温搅拌过夜。反应结束后，冰浴下缓慢加入20％氢氧化钠溶液调节pH至中性，然后用二氯甲烷萃取，有机层经无水硫酸钠干燥后减压浓缩，柱层析分离纯化(石油醚：乙酸乙酯＝1:2)得到白色固体中间体5c(2.87g,61％)。中间体5d的合成由原料5,7-二甲氧基茚酮(5.11g,26.6mmol)按上述方法合成得到白色固体(3.47g,63％)。
(2)中间体6a-6d的合成：中间体5c(1.77g,10mmol)溶于干燥的四氢呋喃(50mL)中，0℃下缓慢加入1mol/L氢化铝锂的四氢呋喃悬液(20mL)，回流反应4小时。反应结束后，冰浴下用30％氢氧化钠溶液淬灭反应，硅藻土过滤，并用甲醇洗涤，滤液经减压浓缩后，再经柱层析纯化(乙酸乙酯：甲醇＝10:1)得到白色油状物6c(0.83g,51％)。中间体6d的合成由中间体5d(2.07g,10mmol)按上述方法合成得到白色油状物(1.04g,54％)。
(3)中间体7a-7i的合成：向耐压管中加入1,2,3,4-四氢异喹啉衍生物(30mmol)、3-碘苯甲酸乙酯(5.52g,20mmol)、碳酸钾(8.34g,60mmol)、碘化亚铜(0.8g,4mmol)和L-脯氨酸(0.92g,8mmol)，再加入二甲基亚砜(25mL)作为反应溶剂，氮气保护，油浴加热到80℃，磁力搅拌反应24小时。反应结束后，向反应体系中加入适量冰水混合物，然后用乙酸乙酯萃取(3×150mL)，有机层经减压浓缩后，残液经柱层析分离纯化(石油醚:乙酸乙酯＝5:1)得到7a-7i。
中间体7b的合成由6-氯-1,2,3,4-四氢异喹啉(5.03g,30mmol)和3-碘苯甲酸乙酯(5.52g,20mmol)按上述方法合成得到白色油状物(3.72g,59％)。
中间体7d的合成由1,2,3,4-四氢异喹啉-6-醇(4.48g,30mmol)和3-碘苯甲酸乙酯(5.52g,20mmol)按上述方法合成得到白色油状物(3.33g,56％)。
中间体7e的合成由7-甲基-1,2,3,4-四氢-异喹啉(4.42g,30mmol)和3-碘苯甲酸乙酯(5.52g,20mmol)按上述方法合成得到白色油状物(3.25g,55％)。
中间体7f的合成由中间体6c(4.90g,30mmol)和3-碘苯甲酸乙酯(5.52g,20mmol)按上述方法合成得到白色油状物(3.86g,62％)。
中间体7g的合成由1,2,3,4-四氢异喹啉-8-醇(4.48g,30mmol)和3-碘苯甲酸乙酯(5.52g,20mmol)按上述方法合成得到白色油状物(3.98g,67％)。
中间体7h的合成由中间体6d(5.80g,30mmol)和3-碘苯甲酸乙酯(5.52g,20mmol)按上述方法合成得到白色油状物(3.68g,54％)。
中间体7i的合成由6,7-二甲氧基-1,2,3,4-四氢异喹啉(5.80g,30mmol)和3-碘苯甲酸乙酯(5.52g,20mmol)按上述方法合成得到白色油状物(4.09g,60％)。
(4)终产物9a-9k的合成：向50mL四氢呋喃中加入中间体7a-7i(10mmol)，再将氢氧化锂(1.19g,50mmol)用乙醇：水(5:1，5mL)溶解后加入，90℃反应3小时。反应结束后，将反应液在真空下旋干，加入少量冰水溶解，然后冰浴下用稀盐酸缓慢调节pH至中性，直至有固体析出。将析出的固体抽滤，干燥，得到中间体8a-8i。8a-8i与2a按上述4a-4h的合成通法进行反应，得到终产物9a-9i。9j和9k分别 由9h和9i在三溴化硼条件下脱甲基得到，即：将9h或9i(0.12mmol)加入干燥的二氯甲烷(15mL)中，在氮气保护下于-20℃逐滴加入三溴化硼(0.11mL,1.2mmol)，室温下反应4小时。反应结束后，冰浴下缓慢加入饱和碳酸氢钠溶液，调节pH至中性。然后用乙酸乙酯萃取，有机层经减压浓缩后，残液经柱层析分离纯化(二氯甲烷:甲醇＝6:1)得到终产物。
化合物9a-9k的结构、外观和核磁共振谱图数据如以下所示。
Figure PCTCN2021136185-appb-000048
9a由8a和2a按上述方法合成，黄色固体(48％)。 1H NMR(400MHz,CD 3OD)δ7.65(t,J＝1.8Hz,1H),7.42(d,J＝8.8Hz,1H),7.37(m,1H),7.31(t,J＝7.9Hz,1H),7.15(dd,J＝9.4,5.6Hz,1H),7.07-7.03(m,2H),6.98(dd,J＝8.8,2.2Hz,1H),6.87-6.80(m,2H),4.39(s,2H),3.57(t,J＝6.0Hz,2H),2.94(m,6H),2.85(s,4H),2.48(s,3H). 13C NMR(100MHz,CD 3OD)δ162.8(d,J＝242.8Hz),153.7,152.3,149.1,138.4(d,J＝7.7Hz),131.7,131.4(d,J＝2.6Hz),130.9(2C),130.6,129.3(d,J＝8.1Hz),117.9,117.7,116.7,115.6(d,J＝21.1Hz),114.1(2C),114.0(d,J＝21.8Hz),102.7,55.7(2C),51.0,50.9,47.3,45.1,43.5,30.0.HRMS(ESI):calcd for C 27H 28N 5F[M+H] +442.2402,found 442.2412.
Figure PCTCN2021136185-appb-000049
9b由8b和2a按上述方法合成，黄色固体(50％)。 1H NMR(400MHz,CD 3OD)δ7.71(s,1H),7.52(d,J＝8.8Hz,1H),7.46(d,J＝7.6Hz,1H),7.38(t,J＝8.0Hz,1H),7.32-7.26(m,1H),7.18-7.14(m,3H),7.11(dd,J＝8.2,2.2Hz,1H),7.05(dd,J＝8.8,1.7Hz,1H),4.42(s,2H),3.61(t,J＝5.8Hz,2H),3.34(s,4H),3.14(s,4H),2.97(t,J＝5.7Hz,2H),2.73(s,3H). 13C NMR(125MHz,CD 3OD)δ159.7,156.5,152.4,146.6,142.9,140.6,140.1,139.1,138.0,137.5,135.4,134.9,133.1,128.2,125.6,125.3,121.6,119.8,109.0,63.2(2C),58.7,58.2(2C),54.7,53.5,37.4.HRMS(ESI):calcd for C 27H 28N 5Cl[M+H] +458.2106,found 458.2098.
Figure PCTCN2021136185-appb-000050
9c由8c和2a按上述方法合成，黄色固体(52％)。 1H NMR(400MHz,CD 3OD)δ7.75(s,1H),7.55(d,J＝8.8Hz,1H),7.49-7.39(m,2H),7.21(d,J＝2.0Hz,1H),7.16(d,J＝8.2Hz,2H),7.11(dd,J＝8.8,2.2Hz,1H),6.82-6.75(m,2H),4.46(s,2H),3.80(s,3H),3.67(t,J＝5.9Hz,2H),3.41(brs,4H),3.29-3.24(m,4H),3.02(t,J＝5.8Hz,2H),2.84(s,3H). 13C NMR(100MHz,DMSO-d 6)δ157.9,157.7,150.4,146.9,135.9,132.2,131.0,129.6,127.6,126.3,124.4,121.1,119.1,115.7,113.0,112.3,111.8,106.9,104.5,56.0,55.0(2C),53.6,49.1,48.6,48.6,45.5,28.4.HRMS(ESI):calcd for C 28H 31N 5O[M+H] +454.2601,found 454.2604.
Figure PCTCN2021136185-appb-000051
9d由8d和2a按上述方法合成，黄色固体(47％)。 1H NMR(500MHz,CD 3OD)δ7.70(s,1H),7.49(d,J＝8.8Hz,1H),7.43(d,J＝7.6Hz,1H),7.37(t,J＝7.9Hz,1H),7.14-7.08(m,2H),7.06-7.01(m,2H),6.63(dd,J＝8.3,2.1Hz,1H),6.60(s,1H),4.38(s,2H),3.60(t,J＝5.9Hz,2H),3.25(t,J＝3.9Hz,4H),2.93(t,J＝5.7Hz,2H),2.79(t,J＝4.1Hz,4H),2.45(s,3H). 13C NMR(125MHz,DMSO-d 6)δ155.8,151.1,150.4,147.8,135.7,131.1,129.5,127.5,124.5,123.2,122.7,118.2,115.6,115.4,114.6,113.9,113.4,111.7,111.2,54.8(2C),49.8(2C),49.2,45.7,45.5,28.4.HRMS(ESI):calcd for C 27H 29N 5O[M+H] +440.2445,found 440.2458.
Figure PCTCN2021136185-appb-000052
9e由8e和2a按上述方法合成，黄色固体(58％)。 1H NMR(400MHz,CD 3OD)δ7.74(s,1H),7.53(d,J＝8.8Hz,1H),7.47(d,J＝7.6Hz,1H),7.40(t,J＝7.9Hz,1H),7.32-7.26(m,1H),7.17(d,J＝1.5Hz,1H),7.13(dd,J＝8.2,1.9Hz,1H),7.09-7.05(m,1H),7.04-6.97(m,2H),4.44(s,2H),3.63(t,J＝5.8Hz,2H),3.38-3.34(m,4H),3.11(t,J＝4.4Hz,4H),2.95(t,J＝5.8Hz,2H),2.71(s,3H),2.31(s,3H). 13C NMR(125MHz,CDCl 3)δ152.2,150.8,147.7,135.6,133.9,131.7,130.1,129.9,128.2,127.2,127.1,125.1,124.4,118.6,116.3,116.2,115.2,112.6,111.1,54.5(2C),50.0,49.5(2C),46.0,45.0,28.6,21.1.HRMS(ESI):calcd for C 28H 31N 5[M+H] +438.2652,found 438.2662.
Figure PCTCN2021136185-appb-000053
9f由8f和2a按上述方法合成，黄色固体(53％)。 1H NMR(400MHz,CDCl 3)δ7.84(s,1H),7.49(d,J＝7.7Hz,2H),7.29(t,J＝8.0Hz,1H),7.09(t,J＝7.9Hz,1H),7.04-6.87(m,2H),6.90(d,J＝8.7Hz,1H),6.71-6.63(m,2H),4.31(s,2H),3.77(s,3H),3.40(t,J＝5.5Hz,2H),3.12(t,J＝4.0Hz,4H),2.78(t,J＝5.2Hz,2H),2.58(t,J＝4.0Hz,4H),2.33(s,3H). 13C NMR(100MHz,CDCl 3)δ156.1,152.3,151.2,147.9,136.1,133.7,132.5,130.8,129.9,129.9,126.9,122.9,120.8,116.5,116.4,115.2,113.1,107.3,101.9,56.0,55.3(2C),54.9(2C),50.2,45.9,45.7,29.2.HRMS(ESI):calcd for C 28H 31N 5O[M+H] +454.2601,found 454.2602.
Figure PCTCN2021136185-appb-000054
9g由8g和2a按上述方法合成，黄色固体(49％)。 1H NMR(500MHz,CD 3OD)δ8.22(s,1H),7.97(d,J＝8.8Hz,1H),7.93(d,J＝7.7Hz,1H),7.84(t,J＝7.7Hz,1H),7.63-7.57(m,2H),7.51-7.44(m,2H),7.19(d,J＝7.7Hz,1H),7.13(d,J＝8.0Hz,1H),4.91(s,2H),4.11(t,J＝6.0Hz,2H),3.69(t,J＝4.5Hz,4H),3.38(t,J＝6.0Hz,2H),3.12(t,J＝4.5Hz,4H),2.83(s,3H). 13C NMR(125MHz,DMSO-d 6)δ155.7,151.0,150.4,147.6,135.6,131.0,129.4,127.3,124.4,123.2,122.7,118.1,115.7,115.5,114.7,113.9,113.3,111.8,111.1,54.4(2C),50.0(2C),49.4,45.7,45.6,28.6.HRMS(ESI):calcd for C 27H 29N 5O[M+H] +440.2445,found 440.2473.
Figure PCTCN2021136185-appb-000055
9h由8h和2a按上述方法合成，黄色固体(50％)。 1H NMR(400MHz,DMSO-d 6)δ7.78(s,1H),7.55(d,J＝6.0Hz,1H),7.46(d,J＝8.5Hz,1H),7.35(t,J＝7.7Hz,1H),7.30-7.18(m,1H),7.07-6.92(m,2H),6.44(s,1H),6.38(s,1H),4.25(s,2H),3.83(s,3H),3.74(s,3H),3.57(s,2H),3.19(s,4H),2.90(s,2H),2.71(s,4H),2.38(s,3H). 13C NMR(125MHz,DMSO-d 6)δ158.7,156.7,150.7,147.4,142.9,136.4,131.1,129.6,127.6,124.4,123.3,118.5,116.0,115.7,114.6,112.0,110.8,104.3,96.2,55.4(2C),55.2(2C),54.3(2C),49.3,45.2,44.8,28.9.HRMS(ESI):calcd for C 29H 33N 5O 2[M+H] +484.2707,found 484.2706.
Figure PCTCN2021136185-appb-000056
9i由8i和2a按上述方法合成，黄色固体(51％)。 1H NMR(400MHz,CD 3OD)δ7.65(s,1H),7.43(d,J＝8.8Hz,1H),7.37-7.33(m,1H),7.30(t,J＝7.9Hz,1H),7.08-7.03(m,2H),6.98(dd,J＝8.8,2.2Hz,1H),6.73(s,1H),6.67(s,1H),4.33(s,2H),3.74(s,3H),3.71(s,3H),3.56(t,J＝5.8Hz,2H),3.27-3.22(m,4H),2.95-2.90(m,4H),2.85(t,J＝5.8Hz,2H),2.54(s,3H). 13C NMR(100MHz,CD 3OD)δ153.8,152.5,149.2,149.1,149.1,131.7,130.9,130.9,128.2,127.8,127.7,118.1,117.7,116.7,114.3,113.1,111.3,103.0,95.3,56.6,56.5,55.7(2C),51.3,51.0(2C),47.9,43.5,29.4.HRMS(ESI):calcd for C 29H 33N 5O 2[M+H] +484.2707,found 484.2727.
Figure PCTCN2021136185-appb-000057
9j：黄色固体(73％)。 1H NMR(400MHz,CD 3OD)δ7.75(s,1H),7.53(d,J＝8.8Hz,1H),7.47(d,J＝7.7Hz,1H),7.41(t,J＝7.9Hz,1H),7.18(d,J＝2.0Hz,1H),7.14(dd,J＝8.2,1.8Hz,1H),7.07(dd,J＝8.8,2.2Hz, 1H),6.23(d,J＝2.2Hz,1H),6.18(d,J＝2.1Hz,1H),4.32(s,2H),3.60(t,J＝5.7Hz,2H),3.37(t,J＝4.4Hz,4H),3.14(t,J＝4.8Hz,4H),2.90(t,J＝5.6Hz,2H),2.73(s,3H). 13C NMR(125MHz,CD 3OD)δ157.3,155.9,153.8,152.8,148.7,140.1,137.8,136.0,131.5,130.8,117.8,117.6,116.7,116.6,114.1,113.9,107.2,103.0,101.3,55.5(2C),50.4(2C),47.4,46.9,44.6,30.3.HRMS(ESI):calcd for C 27H 29N 5O 2[M+H] +456.2394,found 456.2433.
Figure PCTCN2021136185-appb-000058
9k：黄色固体(75％)。 1H NMR(400MHz,DMSO-d 6)δ7.72(s,1H),7.50(d,J＝7.4Hz,2H),7.36(t,J＝7.9Hz,1H),7.06(d,J＝7.5Hz,1H),7.01-6.89(m,2H),6.64(s,1H),6.56(s,1H),4.32(s,2H),3.58(t,J＝5.8Hz,2H),3.15(brs,4H),2.79(t,J＝5.7Hz,2H),2.27(s,4H),1.92(s,3H). 13C NMR(100MHz,DMSO-d 6)δ151.1,150.5,147.8,143.9,143.7,131.1,129.7,129.5,124.8,124.5,115.6,115.5,115.2,114.0,114.0,113.9,113.5,111.8,54.8(2C),49.9,49.3(2C),45.9,45.7,27.5.HRMS(ESI):calcd for C 27H 29N 5O 2[M+H] +456.2394,found 456.2421.
按反应式3所示的合成路线进行化合物14a-14d的合成。以1,2,3,4-四氢异喹啉-6-醇为起始原料，经5步得到14a-14d。
Figure PCTCN2021136185-appb-000059
反应式3中：(a)二碳酸二叔丁酯，4-二甲氨基吡啶，四氢呋喃，室温，0.5h；(b)氢化钠，乙腈，60℃，24h；(c)1.盐酸，1,4-二氧六环，室温，3h；2.碳酸钾，碘化亚铜，L-脯氨酸，二甲基亚砜，80℃，24h，氮气保护；(d)氢氧化锂，四氢呋喃，90℃，3h；(e)1.O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯，N，N-二异丙基乙胺，N,N-二甲基甲酰胺，室温，6h；2.冰乙酸，回流，过夜。
具体步骤如下：
(1)中间体10a的合成：将1,2,3,4-四氢异喹啉-6-醇(2.0g,13.4mmol)和4-二甲氨基吡啶(24mg)加入到四氢呋喃(60mL)中，二碳酸二叔丁酯(3.07mL,13.4mmol)在冰浴下逐滴加入，然后室温搅拌半小时。反应结束后，减压旋干溶剂，加入二氯甲烷稀释，接着用饱和碳酸氢钠溶液洗涤，有机层经无水硫酸钠干燥后减压浓缩，柱层析分离纯化(石油醚：乙酸乙酯＝5:1)得到白色油状物10a(2.34g,70％)。
(2)中间体11a-11d的合成：将中间体10a(373.5mg,1.5mmol)溶于乙腈(10mL)中，冰浴下加入氢化钠(60％，分散在矿物油中，72.9mg,1.8mmol)，室温下搅拌1小时后，加入相应的芳香溴代物(1.8 mmol)，加热到60℃反应24小时。反应结束后，向反应液中加水稀释，然后用乙酸乙酯萃取，有机层经无水硫酸钠干燥后减压浓缩，柱层析分离纯化(石油醚：乙酸乙酯＝10:1)得到中间体11a-11d。
中间体11a由10a和溴乙烷(135μL,1.8mmol)按上述方法合成，得到白色油状物(0.33g,79％)。
中间体11b由10a和正丙基溴(164μL,1.8mmol)按上述方法合成，得到白色油状物(0.31g,72％)。
中间体11c由10a和2-溴丙烷(169μL,1.8mmol)按上述方法合成，得到黄色油状物(0.28g,65％)。
中间体11d由10a和3-溴丙炔(141μL,1.8mmol)按上述方法合成，得到白色油状物(0.31g,71％)。
(3)中间体12a-12d的合成：将Boc衍生物11a-11d(1mmol)加入到1,4-二氧六环(5mL)中，再加入4M盐酸溶液(1.25mL，溶于1,4-二氧六环)，室温下反应3小时后，减压旋干得到的粗产物直接用于下一步反应。将该粗产物加入到耐压管中，再加入碳酸钾(553mg,4mmol)、碘化亚铜(38mg,0.2mmol)、L-脯氨酸(46mg,0.4mmol)、3-碘苯甲酸乙酯(276mg,1mmol)和二甲基亚砜(10mL)，氮气保护，油浴加热到80℃，磁力搅拌反应24小时。反应结束后，向反应体系中加入适量冰水混合物，然后用乙酸乙酯萃取，有机层经减压浓缩后，残液经柱层析分离纯化(石油醚:乙酸乙酯＝10:1)得到12a-12d。
中间体12a由11a和3-碘苯甲酸乙酯按上述方法合成，得到黄色油状物(0.21g,65％)。
中间体12b由11b和3-碘苯甲酸乙酯按上述方法合成，得到黄色油状物(0.23g,67％)。
中间体12c由11c和3-碘苯甲酸乙酯按上述方法合成，得到黄色油状物(0.19g,55％)。
中间体12d由11d和3-碘苯甲酸乙酯按上述方法合成，得到黄色油状物(0.20g,61％)。
(4)终产物14a-14d的合成：14a-14d的合成按9a-9i的合成方法进行。
化合物14a-14d的结构、外观和核磁共振谱图数据如以下所示。
Figure PCTCN2021136185-appb-000060
14a由13a和2a按上述方法合成，黄色固体(40％)。 1H NMR(400MHz,DMSO-d 6)δ7.80(s,1H),7.53(d,J＝7.3Hz,1H),7.46(d,J＝8.6Hz,1H),7.32(t,J＝7.8Hz,1H),7.15(d,J＝7.9Hz,1H),7.08-6.92(m,3H),6.76-6.66(m,2H),4.41(s,2H),3.97(q,J＝7.0Hz,2H),3.39-3.24(m,6H),2.97-2.87(m,6H),2.53(s,3H),1.28(t,J＝6.7Hz,3H). 13C NMR(125MHz,DMSO-d 6)δ157.1,151.4,150.5,147.1,136.0,131.1,129.7,127.8,126.4,124.5,123.5,118.5,115.9,115.7,114.2,113.7,112.9,112.1,111.1,63.1,53.8(2C),49.3,48.7(2C),45.6,44.1,28.5,14.9.HRMS(ESI):calcd for C 29H 33N 5O[M+H] +468.2758,found 468.2767.
Figure PCTCN2021136185-appb-000061
14b由13b和2a按上述方法合成，黄色固体(41％)。 1H NMR(400MHz,CD 3OD)δ7.74(s,1H),7.54(d,J＝8.7Hz,1H),7.47(d,J＝7.4Hz,1H),7.41(t,J＝7.9Hz,1H),7.18(s,1H),7.14(dd,J＝8.3,3.6Hz,2H),7.08(dd,J＝8.8,2.0Hz,1H),6.79-6.73(m,2H),4.44(s,2H),3.92(t,J＝6.5Hz,2H),3.64(t,J＝5.8Hz,2H), 3.38-3.33(m,4H),3.07-2.97(m,6H),2.66(s,3H),1.82-1.74(m,2H),1.05(t,J＝7.4Hz,3H). 13C NMR(125MHz,CD 3OD)δ163.7,162.1,157.6,150.8,136.3,131.5,130.5,130.1,130.0,128.0,126.7,122.2,116.2,115.9,114.1,113.8,113.2,112.9,112.3,69.3,54.9(2C),49.9,49.9,49.6,45.9,45.6,28.9,22.5,10.9.HRMS(ESI):calcd for C 30H 35N 5O[M+H] +482.2914,found 482.2921.
Figure PCTCN2021136185-appb-000062
14c由13c和2a按上述方法合成，黄色固体(37％)。 1H NMR(400MHz,CD 3OD)δ7.71(s,1H),7.51(d,J＝8.7Hz,1H),7.45(d,J＝7.6Hz,1H),7.37(t,J＝7.9Hz,1H),7.29-7.25(m,1H),7.13(s,1H),7.10-7.06(m,1H),7.03(dd,J＝8.8,2.0Hz,1H),6.76-6.65(m,2H),4.58-4.49(m,1H),4.38(s,2H),3.59(t,J＝5.5Hz,2H),3.36-3.32(m,2H),3.30-3.26(m,2H),3.11-3.05(m,4H),2.94(t,J＝5.7Hz,2H),2.68(s,3H),1.28(d,J＝6.0Hz,6H). 13C NMR(125MHz,CD 3OD)δ156.4,152.3,151.0,147.5,135.8,130.2,129.5,127.5,127.2,126.2,124.0,117.4,116.4,116.1,115.1,114.0,112.5,111.0,101.4,69.6,54.1(2C),49.5,49.2(2C),46.1,43.4,28.8,21.0(2C).HRMS(ESI):calcd for C 30H 35N 5O[M+H] +482.2914,found 482.2926.
Figure PCTCN2021136185-appb-000063
14d由13d和2a按上述方法合成，黄色固体(39％)。 1H NMR(400MHz,DMSO-d 6)δ7.74(s,1H),7.51(d,J＝7.6Hz,1H),7.46(d,J＝8.8Hz,1H),7.41-7.35(m,1H),7.20(d,J＝8.1Hz,1H),7.07(dd,J＝8.2,2.0Hz,1H),7.02(s,1H),6.96(dd,J＝8.8,2.2Hz,1H),6.88-6.80(m,2H),4.77(d,J＝2.3Hz,2H),4.42(s,2H),3.61(t,J＝5.6Hz,2H),3.54(t,J＝2.3Hz,1H),3.23-3.15(m,4H),2.94(t,J＝5.6Hz,2H),2.81-2.71(m,4H),2.44(s,3H). 13C NMR(125MHz,DMSO-d 6)δ156.1,151.7,150.8,147.7,136.4,131.5,130.0,128.1,127.6,125.8,124.1,119.1,116.2,116.1,114.6,114.6,113.5,112.2,110.7,79.9,78.6,55.8,54.5(2C),49.6,49.5(2C),45.9,45.0,28.9.HRMS(ESI):calcd for C 30H 31N 5O[M+H] +478.2601,found 478.2577.
按反应式4所示的合成路线进行化合物17a-17c的合成。3-碘苯甲酸乙酯或3-乙氧羰基苯硼酸经偶联反应得到中间体15a-15c，然后15a-15c在碱性条件下水解得到中间体16a-16c，接着与中间体邻苯二胺衍生物2a通过酰胺反应和关环反应，最终得到终产物17a-17c。
Figure PCTCN2021136185-appb-000064
反应式4中：(a)碳酸钾，碘化亚铜，L-脯氨酸，二甲基亚砜，80℃，24h；或者碳酸钠，四三苯基磷钯，1,4-二氧六环/水，80℃，16h，氮气保护；(b)氢氧化锂，四氢呋喃，90℃，3h；(c)1.O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯，N，N-二异丙基乙胺，N,N-二甲基甲酰胺，室温，6h；2.冰乙酸，回流，过夜。
具体步骤如下：
(1)中间体15a-15c的合成：1-(2-嘧啶基)哌嗪或吗啉与3-碘苯甲酸乙酯的反应按7a-7i的合成条件进行，分别得到中间体15a(白色油状物，73％)和15b(白色油状物，78％)。对于15c的合成，将4-溴-1,2-亚甲二氧基苯(400mg,2mmol)和3-乙氧羰基苯硼酸(388mg,2mmol)加入到1,4-二氧六环/水(4:1,15mL)的混合溶剂中，接着加入碳酸钠(424mg,4mmol)和四三苯基磷钯(115.5mg,0.1mmol)，氮气保护下，油浴加热到80℃，磁力搅拌反应16小时。反应结束后，冷却至室温，过滤，滤液经1N氢氧化钠溶液洗涤后，用二氯甲烷萃取，有机层经无水硫酸钠干燥后减压浓缩，柱层析分离纯化(石油醚:乙酸乙酯＝20:1)得到15c(白色油状物，76％)。
(2)终产物17a-17c的合成：17a-17c的合成按9a-9i的合成方法进行。
化合物17a-17c的结构、外观和核磁共振谱图数据如以下所示。
Figure PCTCN2021136185-appb-000065
17a由16a和2a按上述方法合成，白色固体(45％)。 1H NMR(400MHz,CD 3OD)δ8.33(d,J＝4.8Hz,2H),7.69(s,1H),7.53-7.47(m,2H),7.37(t,J＝7.8Hz,1H),7.13-7.08(m,2H),7.03(dd,J＝8.8,2.1Hz,1H),6.59(t,J＝4.8Hz,1H),3.95(t,J＝5.2Hz,4H),3.34-3.30(m,4H),3.24(t,J＝4.8Hz,4H),2.77(t,J＝4.8Hz,4H),2.44(s,3H). 13C NMR(100MHz,DMSO-d 6)δ161.7,158.5(3C),151.8,148.2,131.6(2C),130.0(2C),117.5,117.2,113.6(2C),110.9(3C),55.1(2C),50.1,48.6(2C),45.9,43.7(2C),41.8.HRMS(ESI):calcd for C 26H 30N 8[M+H] +455.2666,found 455.2701.
Figure PCTCN2021136185-appb-000066
17b由16b和2a按上述方法合成，白色固体(40％)。 1H NMR(400MHz,CD 3OD)δ7.64(s,1H),7.53-7.43(m,2H),7.34(t,J＝8.0Hz,1H),7.09(s,1H),7.00(d,J＝8.7Hz,2H),3.79(t,J＝4.8Hz,4H),3.20-3.13(m,8H),2.64(t,J＝4.8Hz,4H),2.33(s,3H). 13C NMR(100MHz,DMSO-d 6)δ151.4,150.8,147.8,131.1,129.5,129.1,118.0,117.0,116.1,113.9,113.7,112.4,110.7,66.1(2C),54.8(2C),49.9(2C),48.4(2C),45.7.HRMS(ESI):calcd for C 22H 27N 5O[M+H] +378.2288,found 378.2289.
Figure PCTCN2021136185-appb-000067
17c由16c和2a按上述方法合成，棕褐色固体(44％)。 1H NMR(400MHz,CD 3OD)δ8.24(s,1H),7.95(d,J＝7.7Hz,1H),7.62(d,J＝7.8Hz,1H),7.54-7.49(m,2H),7.19(s,1H),7.18-7.16(m,1H),7.11(s,1H),7.04(dd,J＝8.8,2.0Hz,1H),6.90(d,J＝7.8Hz,1H),5.99(s,2H),3.21(t,J＝4.8Hz,4H),2.67(t,J＝4.8Hz,4H),2.37(s,3H). 13C NMR(100MHz,CD 3OD)δ152.7,149.7,149.7,148.9,142.9,135.8,131.5,130.5,129.1,125.8,125.7,121.7,116.6,109.5,108.3,102.6,56.1(2C),51.6(2C),46.0.HRMS(ESI):calcd for C 25H 24N 4O 2[M+H] +413.1972,found 413.1998.
为研究化合物在细胞内的靶点，我们在炔基化合物14d的结构上连接了生物素，合成得到了探针Biotin-14d，具体反应步骤如下：将Biotin-PEG 3-N 3(22mg，0.05mmol)与14d(24mg，0.05mmol)加入到甲醇/水(4:1，2.5mL)混合溶液中，接着加入1M五水硫酸铜溶液(20μL)和新鲜配制的1M抗坏血酸钠溶液(20μL)，氮气保护，室温下反应过夜。反应结束后，减压浓缩，粗产物经制备高效液相色谱梯度洗脱，纯化得到Biotin-14d。流动相为含10％-75％乙腈的0.1％甲酸水，30分钟，流速为3mL/min。HRMS(ESI):calcd for C 48H 63N 11O 6S[M+2H] 2+461.7415,found 461.7418.
Figure PCTCN2021136185-appb-000068
实施例2实施例1所得的化合物的铁死亡抑制活性测试
(1)细胞培养：HT22细胞(小鼠海马神经元细胞)使用含有10％胎牛血清、1％双抗的DMEM高糖培养基，在37℃，5％二氧化碳浓度条件下进行常规培养和传代。
(2)药物干预：细胞按3000个/孔接种于96孔板中，培养24小时后吸除原培养液，加入1μM Erastin±化合物作用24小时。
(3)检测：用Cell Counting Kit-8(CCK-8)试剂盒检测细胞存活率，每孔加入10μl CCK-8溶液，37℃下孵育4小时后，在450nm波长下检测吸光度OD值。
(4)数据处理：按如下公式计算：细胞存活率(％)＝(OD 样品-OD 空白)/(OD 对照-OD 空白)×100％，以细胞存活率为纵坐标，化合物浓度为横坐标绘制各化合物在Erastin诱导铁死亡条件下细胞的存活曲线图，并计算各化合物的EC 50值以评价各化合物对Erastin诱导铁死亡的抑制活性。
化合物对Erastin诱导铁死亡的抑制活性如表1所示。其中，Ferrostatin-1(Fer-1)和deferoxamine(DFO)是已知的铁死亡抑制剂，在此实验中作为阳性药使用。除了化合物4g-4h、9d、9h、9j-9k和17a-17c的活性较差外，其他化合物的EC 50值均是μM级别，并且化合物9a是所有化合物中活性最好的，EC 50＝0.29μM。因此，我们对化合物9a的铁死亡抑制活性进行了更加深入的评价。
表1化合物在HT22细胞上对Erastin诱导铁死亡的抑制活性
Figure PCTCN2021136185-appb-000069
n.d.:1.0μM化合物和Erastin共同处理细胞时，细胞存活率在小于50％。
实施例3实施例1所得的化合物9a的铁死亡抑制活性评价
为更加深入地评价化合物9a的铁死亡抑制活性，分别检测了在HT22细胞上，化合物9a对GPX4共价抑制剂RSL3诱导铁死亡的抑制活性；在HT1080细胞上，化合物9a对敲除GPX4诱导铁死亡的抑制活性。并且检测了化合物9a对铁死亡的两个特征(RSL3诱导的脂质过氧化和PTGS2 mRNA上调)的抑制效果，结果如图1所示。
(1)RSL3诱导铁死亡
HT22细胞按3000个/孔接种于96孔板中，培养24小时后吸除原培养液，加入1μM RSL3±化合物作用24小时后，每孔加入10μl CCK-8溶液，37℃下孵育4小时后，在450nm波长下检测吸光度OD值。
(2)敲除GPX4诱导铁死亡
HT1080细胞(人纤维肉瘤细胞)使用含有10％胎牛血清、1％双抗的MEM培养基，在37℃，5％二氧化碳浓度条件下进行常规培养和传代。将50nM的siRNA scramble和siRNA GPX4分别用Lipofectamine TM3000试剂转入HT1080细胞，12小时后加入化合物，继续孵育36小时后收获细胞进行qPCR和CCK-8实验。qPCR实验具体步骤如下：待处理的细胞用PBS洗两次，用RNAiso plus试剂提取总RNA，使用ReverTra Ace qPCR RT Master Mix将1μg总RNA逆转录为cDNA，再使用SYBR Green Realtime PCR Master Mix进行PCR扩增。
(3)检测脂质ROS
流式检测：HT22细胞按3×10 4个/孔接种于6孔板中培养24小时后，加入1μM RSL3±化合物作用3小时，然后用5μM BODIPY-C11探针在37℃下孵育半小时，细胞用PBS洗一次后，用胰酶消化、离心并收集在1ml PBS中，然后用流式在488nm下检测FL1通道的荧光强度。每个样品至少检测10,000个细胞。
成像检测：HT22细胞按3000个/孔接种于96孔板中培养24小时后，加入1μM RSL3±化合物作用3小时，然后用5μM BODIPY-C11探针和Hoechst 33342(1μg/ml)在37℃下共同孵育半小时，细胞用PBS洗一次后，直接用FV3000激光共聚焦显微镜拍照(60×)。
(4)检测PTGS2 mRNA水平
HT22细胞按3×10 4个/孔接种于6孔板中培养24小时后，加入1μM RSL3±化合物作用12小时，用PBS洗两次，用RNAiso plus试剂提取总RNA，使用ReverTra Ace qPCR RT Master Mix将1μg总RNA逆转录为cDNA，再使用SYBR Green Realtime PCR Master Mix进行PCR扩增。
如图1所示，与阳性化合物Fer-1和DFO类似，化合物9a剂量依赖性地抑制了RSL3诱导的HT22细胞铁死亡；在敲除GPX4诱导HT1080细胞铁死亡的模型上，化合物9a同样具有抑制活性；活细胞成像结果显示，化合物9a能够显著抑制RSL3诱导的脂质ROS增加，与流式的结果一致，此外化合物9a对RSL3诱导的胞质ROS的增加也具有抑制活性；最后，化合物9a也浓度依赖性地抑制了RSL3诱导的PTGS2 mRNA上调。
实施例4实施例1所得的化合物9a在体内对缺血再灌注损伤的缓解作用
缺血性中风是中风最常见的形式，也是导致死亡和永久性残疾的主要疾病之一。到目前为止，尚无有效的治疗方法。研究发现，铁死亡参与了缺血性中风病理过程中神经元的损伤和死亡，抑制铁死亡可以在体内减轻大脑损伤。由于化合物9a在HT22细胞上具有显著的铁死亡抑制活性，所以我们研究了在大脑中动脉阻塞(Middle Cerebral Artery Occlusion，MCAO)模型上，该化合物是否可以保护SD大鼠免受缺血/再灌注造成的神经损伤。
(1)试验动物分组
将动物随机分为4组，分别为空白对照组、模型对照组、阳性对照组、受试样品9a组，每组12只。给药剂量：受试样品9a 10mg/kg，阳性对照药依达拉奉10mg/kg；空白对照组和模型对照组动物同法给予生理盐水。给药频率：手术前半小时给药一次，术后2小时给药一次；给药途径：腹腔注射给药，体积为5ml/kg。
(2)造模
将大鼠用7％水合氯醛5ml/kg腹腔注射麻醉，颈正中切口，分离、结扎左侧颈总动脉、颈外动脉，并分离翼额动脉。在颈内动脉近端备线、远端放置动脉夹，颈总动脉分叉处切口，插入4－0尼龙线，其深度为17～20mm，栓线进入颈内动脉，入颅至大脑前动脉，阻断大脑中动脉所有血流来源，让其缺血1.5h然后拔出栓线约1cm，实现再灌注。伤口以碘伏消毒，缝合皮肤。然后回笼饲养。空白对照组除不插线外，其余步骤同上。
(3)TTC检测
术后24h进行行为学检测，有明显手术侧(即左侧)Horner症(左侧眼睑下垂，眼球凹陷)及手术侧 (即左侧)偏瘫体症(不能完全伸展左前肢，行走时向左侧倾倒或转圈)即为模型成功，并对各动物模型程度进行评分。行为学检测结束后处死动物，取大脑切片进行TTC染色，白色为脑梗死区域，红色为正常区域，计算动物脑梗死体积。
如图2所示，TTC染色结果表明，相比于模型组，9a处理后脑梗死体积明显降低，行为更加正常，说明化合物9a在体内能够显著缓解缺血/再灌注诱导的大脑损伤，有望发展成为治疗缺血性中风的先导化合物。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。

Claims (10)

  1. 一种苯并咪唑类化合物，其特征在于，具有如式(Ⅰ)或式(Ⅱ)所示结构：
    Figure PCTCN2021136185-appb-100001
    其中，R 1为
    Figure PCTCN2021136185-appb-100002
    R 2为
    Figure PCTCN2021136185-appb-100003
    R为卤素、甲基、烷氧基、炔氧基或羟基；
    X为C或N。
  2. 根据权利要求1所述苯并咪唑类化合物，其特征在于，所述卤素为F或Cl；所述烷氧基为-OCH 3、-OCH 2CH 3、-OCH 2CH 2CH 3或-OCH(CH 3) 2；所述炔氧基为-OCH 2C≡CH。
  3. 根据权利要求1所述苯并咪唑类化合物，其特征在于，所述R在6位、7位或8位中的一位或两位为取代。
  4. 根据权利要求2或3所述苯并咪唑类化合物，其特征在于，R在6位取代时，R为-F、-Cl、-OCH 3、-OH、-OCH 2CH 3、-OCH 2CH 2CH 3、-OCH(CH 3) 2或-OCH 2C≡CH；
    R在7位取代时，R为-CH 3；
    R在8位取代时，R为-OCH 3或-OH；
    R在6位和7位取代时，R为-OH或-OCH 3；
    R在6位和8位取代时，R为-OH或-OCH 3。
  5. 权利要求1～4任一所述苯并咪唑类化合物的制备方法，其特征在于，包括如下步骤：
    S11：2-硝基-5-氯苯胺和环状脂肪胺R 1-H进行偶联反应，得
    Figure PCTCN2021136185-appb-100004
    S12：
    Figure PCTCN2021136185-appb-100005
    在镍和水合肼条件下还原为
    Figure PCTCN2021136185-appb-100006
    S13：
    Figure PCTCN2021136185-appb-100007
    反应，即得式(Ⅰ)所示结构的苯并咪唑类化合物
    Figure PCTCN2021136185-appb-100008
    或当R 2为
    Figure PCTCN2021136185-appb-100009
    时：
    S21：茚酮衍生物
    Figure PCTCN2021136185-appb-100010
    在酸性条件下，与叠氮化钠进行Aube-Schmidt重排反应得
    Figure PCTCN2021136185-appb-100011
    S22：
    Figure PCTCN2021136185-appb-100012
    经还原后得四氢异喹啉衍生物
    Figure PCTCN2021136185-appb-100013
    S23：四氢异喹啉衍生物
    Figure PCTCN2021136185-appb-100014
    和3-碘苯甲酸乙酯
    Figure PCTCN2021136185-appb-100015
    经偶联反应得
    Figure PCTCN2021136185-appb-100016
    S24：
    Figure PCTCN2021136185-appb-100017
    在碱性条件下水解得
    Figure PCTCN2021136185-appb-100018
    S25：
    Figure PCTCN2021136185-appb-100019
    和邻苯二胺衍生物
    Figure PCTCN2021136185-appb-100020
    通过酰胺反应和关环反应 即得式(Ⅱ)所示结构的苯并咪唑类化合物
    Figure PCTCN2021136185-appb-100021
    或当R 2为
    Figure PCTCN2021136185-appb-100022
    时：
    S31：3-碘苯甲酸乙酯
    Figure PCTCN2021136185-appb-100023
    或3-乙氧羰基苯硼酸
    Figure PCTCN2021136185-appb-100024
    与R 2-H或R 2-Br经偶联反应得
    Figure PCTCN2021136185-appb-100025
    S32：
    Figure PCTCN2021136185-appb-100026
    在碱性条件下水解得
    Figure PCTCN2021136185-appb-100027
    S33：
    Figure PCTCN2021136185-appb-100028
    与中间体邻苯二胺衍生物
    Figure PCTCN2021136185-appb-100029
    通过酰胺反应和关环反应，即得式(Ⅱ)所示结构的苯并咪唑类化合物
    Figure PCTCN2021136185-appb-100030
  6. 根据权利要求5所述制备方法，其特征在于，S11中偶联反应的条件为碱性条件下，110℃反应16h；S12中还原的温度为60℃，时间为2h；S13中酰胺反应为常温搅拌6h，后续关环反应为在冰乙酸下，回流反应过夜。
  7. 根据权利要求5所述制备方法，其特征在于，S21中以二氯甲烷和甲基磺酸的混合液作为溶剂；S22中利用氢化铝锂进行还原；S23中偶联反应的条件为：惰性气氛下，80℃下反应24h；S25中酰胺反应和关环反应的条件同S13。
  8. 根据权利要求5所述制备方法，其特征在于，S31中偶联反应的条件为：惰性气氛下，80℃下反应24h或16h；S33中酰胺反应和关环反应的条件同S13。
  9. 权利要求1～4任一所述苯并咪唑类化合物在制备铁死亡抑制剂中的应用。
  10. 根据权利要求9所述应用，其特征在于，所述苯并咪唑类化合物在制备缺血性脑中风药物中的应用。
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