[go: up one dir, main page]

WO2025087033A1 - 一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物及其制备方法和应用 - Google Patents

一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物及其制备方法和应用 Download PDF

Info

Publication number
WO2025087033A1
WO2025087033A1 PCT/CN2024/123539 CN2024123539W WO2025087033A1 WO 2025087033 A1 WO2025087033 A1 WO 2025087033A1 CN 2024123539 W CN2024123539 W CN 2024123539W WO 2025087033 A1 WO2025087033 A1 WO 2025087033A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
thiadiazole
ferroptosis
nmr
benzene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/123539
Other languages
English (en)
French (fr)
Inventor
王勇
邱雪
闫江坤
包罗
范雪静
孔令秀
韩新宇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ocean University of China
Original Assignee
Ocean University of China
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ocean University of China filed Critical Ocean University of China
Publication of WO2025087033A1 publication Critical patent/WO2025087033A1/zh
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
    • C07D285/01—Five-membered rings
    • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
    • C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
    • C07D285/12—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1007—Non-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1011—Condensed systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
    • C09K2211/1051—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with sulfur

Definitions

  • the invention belongs to the field of medical technology, and specifically relates to a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, and a preparation method and application thereof.
  • Apoptosis is the most typical form of regulated cell death, which triggers cell death by activating caspases.
  • Ferroptosis is a new type of cell death discovered in recent years. It is an oxidative cell death induced by various reasons and is iron-dependent. Its occurrence is caused by the imbalance of the generation and degradation of reactive oxygen species (ROS) in intracellular lipids.
  • ROS reactive oxygen species
  • ferroptosis The control mechanism of ferroptosis elucidated in the first few years of the discovery of ferroptosis mainly revolves around cysteine and glutathione metabolism, and phospholipid peroxidase GPX4 prevents the accumulation of peroxidized lipids.
  • Ferroptosis inducers act directly or indirectly on glutathione peroxidase (GPXs) through different pathways, resulting in reduced cellular antioxidant capacity, ROS accumulation, and ultimately cell oxidative death.
  • GPXs glutathione peroxidase
  • the complex interaction between lipid, iron and cysteine metabolism has become an important regulator of this cell death pathway.
  • modulation of ferroptosis has emerged as an attractive strategy for intervention in human diseases, including cancer, neurodegenerative diseases, and ischemic disorders.
  • Ferroptosis can be inhibited by iron chelators, lipophilic antioxidants, and/or ferrostatin-1 (fer-1).
  • Fer-1 is an arylalkylamine with antioxidant properties and was one of the first identified ferroptosis inhibitors. Fer-1 acts as a lipid peroxidation reductant, intercepting and scavenging lipid free radicals via hydrogen atom transfer or direct reduction. However, its short half-life makes it unsuitable for further pharmacological evaluation.
  • fluorescent probes detect substances by increasing or decreasing fluorescence intensity. Based on this, a fluorescent probe that can react with ONOO- is designed. This probe can consume ONOO- through chemical reactions to play an antioxidant role. At the same time, the changes in the fluorescence spectrum caused before and after the reaction enable it to have real-time monitoring functions.
  • This type of antioxidant fluorescent probe with self-indicating function can achieve self-monitoring in the process of reversing ferroptosis without the need for additional processing of experimental samples, which provides a new idea for the development of evaluation methods for ferroptosis antioxidants.
  • the present invention provides a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine and a preparation method and application thereof.
  • the compound has strong antioxidant activity and good metabolic stability, and is suitable for the treatment of ferroptosis-related diseases; and has obvious fluorescence spectrum changes, has extremely strong selectivity for ONOO - , and the phenomenon is obvious and easy to identify.
  • the present invention provides a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, the structural formula of which is shown below:
  • R1 is selected from hydrogen, C1 - C6 alkyl, C1 - C6 alkoxy, C2- C6 alkenyl, C2 - C6 alkynyl, C3 - C8 cycloalkyl, C3 - C8 cycloalkoxy, amino, phenyl, benzyl, naphthyl, C5 - C10 aromatic heterocyclic group or C3 - C7 saturated heterocyclic group ;
  • R 2 is selected from C 0 ⁇ C 8 alkyl, C 3 ⁇ C 12 cycloalkyl, adamantyl or polyalkynyl;
  • R 3 is selected from hydrogen, alkyl, aryl, C 1 -C 6 alkyl-aryl, C 1 -C 6 alkyl-phenol or C 3 ⁇ C 10 cycloalkyl;
  • R 1 is selected from C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkoxy, aryl and derivatives.
  • R 2 is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and derivatives thereof, cycloheptyl, adamantyl and derivatives thereof.
  • R 3 is selected from cyclopropyl, cyclopentyl, cyclohexyl, arylbenzyl and derivatives thereof.
  • n 0, 2-4.
  • the compound is specifically compound I-1, II-1 to II-9, III-1 to III-15, IIII-1 to IIII-12, and its structural formula is as follows:
  • the present invention also provides a method for preparing the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound, which comprises the following steps:
  • step (2) adding the compound 2 prepared in step (1) and Lawesson's reagent to dioxane, reacting at 110° C. overnight, and separating and purifying after the reaction to obtain a thiadiazole compound 3;
  • step (3) adding the thiadiazole compound 3 prepared in step (2) and the corresponding amine to DMSO, reacting at 60° C. for 18 hours, and separating and purifying after the reaction to obtain the corresponding intermediate compound 4;
  • step (3) (4) adding the intermediate compound 4 of step (3) and Pd/C to methanol, stirring at room temperature for 6 hours under H2 conditions, reacting until completion, and then separating and purifying to obtain an amino-containing compound 5;
  • step (1) the molar ratio of 4-chloro-3-nitrobenzoic acid, NH 2 NHBoc, HBTU and DIEPA is 1:1.5:1.5:1.5.
  • the molar ratio of compound 2 to Lawesson's reagent is 1:2.5.
  • step (3) the molar ratio of the thiadiazole compound 3 to the corresponding amine is 1:1.5.
  • the mass ratio of the intermediate compound 4 to Pd/C is 10:1.
  • the molar ratio of the amino compound 5, the corresponding aldehyde and NaBH(OAc) 3 in step (5) is 1:1.1:2.
  • the specific method for separation and purification in step (1) is: after the reaction is completed, the mixed solution is poured into 30 mL of water, the aqueous layer is extracted with ethyl acetate, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain a light yellow solid compound 2.
  • the specific method of separation and purification in step (2) is: after the reaction is completed, cool to room temperature, evaporate the solvent, extract the mixture with water and ethyl acetate, combine the organic layers, and then wash the combined organic layers with 10% sodium bicarbonate solution. Then, dry the organic layer with sodium sulfate, filter, concentrate and separate by column chromatography to obtain thiadiazole compound 3.
  • the specific method for separation and purification in step (3) is: after the reaction is completed, the mixed reaction liquid is poured into 30 mL of water, the aqueous layer is extracted with ethyl acetate, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain the intermediate compound 4.
  • the specific method of separation and purification in step (4) is: after the reaction is completed, the solution is filtered with diatomaceous earth, the solvent is evaporated under vacuum, and the crude product is separated by column chromatography to obtain the amino-containing compound 4;
  • the specific method of separation and purification in step (5) is: after the reaction is completed , the reaction solution is poured into a saturated sodium bicarbonate aqueous solution, the aqueous layer is extracted with CH2Cl2 , the combined organic layer is washed with a saturated sodium bicarbonate aqueous solution, and then washed with brine, dried, filtered, and concentrated in vacuo. The mixture is separated by column chromatography to obtain a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine.
  • the present invention also provides the use of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine-containing compounds in the preparation of ferroptosis inhibitors.
  • the present invention also provides the use of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine-containing compounds in the preparation of drugs for ferroptosis-related diseases.
  • ferroptosis-related diseases include neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, liver and kidney failure, inflammation, and diabetic complications.
  • the drug contains a (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound or a pharmaceutically acceptable salt thereof, an excipient or a carrier.
  • the present invention also provides the use of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine-containing compound in the preparation of antioxidant fluorescent probes or antioxidant indicators.
  • the antioxidant fluorescent probe or antioxidant indicator is used for high-sensitivity detection of ONOO- , which can effectively reverse the process of cell ferroptosis and has self-indication capability.
  • the present invention has the following advantages and beneficial effects:
  • the present invention has designed and obtained a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, which can effectively inhibit ferroptosis and has good metabolic stability, and can be used for the study of ferroptosis-related diseases;
  • the preparation method of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound of the present invention is simple and convenient, with high yield, and is suitable for large-scale promotion and application;
  • the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compounds described in the present invention can be used as the fluorescent probe and have obvious fluorescence spectrum changes.
  • the effect of the monitoring effect can be evaluated in real time through the changes in its own fluorescence spectrum.
  • FIG1A is a hydrogen nuclear magnetic resonance spectrum of compound I-1
  • FIG1B is a carbon spectrum of compound I-1
  • FIG1C is a high-resolution mass spectrum of compound I-1;
  • Fig. 2 is a graph showing the selectivity test results of the antioxidant fluorescent probe I-1 for detecting ONOO - , wherein 1-14 represent the bioactive small molecules Blank, Cu 2+ , Zn 2+ , Fe 3+ , Fe 2+ , Al 3+ , Ca 2+ , SO 4 2- , SO 3 2- , NO 2 - , O 2- , t-BuOOH, H 2 O 2 , HClO, ONOO - , ⁇ OH, Vc - , GSH, and VC, respectively;
  • FIG3 is a fluorescence spectrum result diagram of the antioxidant fluorescent probe I-1 detecting ONOO - ;
  • FIG4 is a graph showing the fluorescence spectra of the antioxidant fluorescent probe I-1 in different solvents
  • FIG5 is a graph showing the results of flow cytometry evaluation of the antioxidant activity of I-1 and its self-indicating ability.
  • compound I-1 was prepared by the experimental method described above.
  • 1 H NMR 400 MHz, Chloroform-d
  • ⁇ 7.39 s, 1H
  • 3.29 m, 1H
  • 2.74 s, 3H
  • 2.09-2.04 m, 2H
  • 1.80-1.64 m, 4H
  • 1.46-1.32 m, 4H.
  • This example uses the experimental method described above to prepare compounds II-1 to II-9:
  • ferroptosis inhibitors such as RSL-3 can induce ferroptosis in cells, which can also be blocked by other small molecules, such as lipophilic antioxidants, such as Ferrostatin-1 (fer-1), Liproxstatin, etc. Therefore, the ability of ferroptosis inhibitors to block ferroptosis can be indicated by the reversal of ferroptosis induced by ferroptosis inducers.
  • Human renal carcinoma cell line OS-RC-2 and human neuroblastoma cell line SH-SY5Y were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.
  • MTT method as follows: Human renal cancer cell line OS-RC-2/human neuroblastoma cell SH-SY5Y in logarithmic growth phase were digested, collected and diluted, and about 4000-5000 cells were seeded in each well of 96-well plate. The experiment set up 3 replicate wells, 80 ⁇ L per well. Incubate overnight in a 37°C, 5% CO 2 incubator. The experiment set up a DMSO control group and nine different concentrations of compound administration groups. Different concentrations of compounds were added to the administration group, and a DMSO control group (the same dilution multiple as the highest concentration compound) was set up.
  • Table 1 Test results of ferroptosis inhibition activity of compounds In the table: "A” indicates IC 50 ⁇ 100nM, “B” indicates IC 50 >100nM and ⁇ 500nM, and “C” indicates IC 50 >500 nM and ⁇ 1 ⁇ M, “D” indicates IC50 >1 ⁇ M and ⁇ 10 ⁇ M.
  • Example 7 In vitro/in vivo drug metabolism properties of compounds
  • Plasma and liver microsomal homogenate samples were stored at -20 °C, taken out before use, and gradually warmed to room temperature.
  • the compound was dissolved in acetonitrile to make a 4 mg/mL stock solution, then added to plasma or liver microsomal homogenate to a final concentration of 0.4 mg/mL and incubated at 37 °C for the desired time (0, 0.25, 0.5, 1, 2, 4, 8, 12, 24 and 24 h).
  • the acetonitrile was terminated at the end point of the incubation, the mixture was vortexed for 30 s and centrifuged at 12000 rpm for 10 min, and then filtered through a 0.22 ⁇ M filter.
  • the filtrate was analyzed by HPLC system (Agilent 1260 HPLC) with Alltima C18 (5 ⁇ m, 4.6 mm ⁇ 250 mm) column.
  • the flow rate was maintained at 400 ⁇ L/min, and the initial flow conditions were 80% solvent A (water containing 0.1% acetic acid) and 20% solvent B (methanol containing 0.1% acetic acid).
  • Solvent B was increased to 80% in 0.50 min and maintained for 1.50 min.
  • solvent B was increased to 100% in 5.00 min and maintained there for 3 min.
  • solvent B was reduced to initial conditions (20%) in 0.50 min.
  • the total running time was 12.00 min.
  • the Fer-1 structure-based thiadiazole compounds of the present invention exhibit good stability in plasma and liver microsomes in vitro, have good ADME properties, and can be used for in vivo biological activity studies.
  • In vivo test method After a single dose of intravenous injection of the compound in male mice, blood samples and brain tissue were collected at 0, 0.5h, 2h, 4h, and 8h. The concentration of the compound in mouse plasma and brain tissue was determined by LC-MS/MS and the relevant pharmacokinetic parameters were calculated to investigate the pharmacokinetic characteristics and brain tissue distribution of the compound in mice. From the results, it can be seen that compound I-1 exhibits good metabolic stability in mice, with a half-life of 2.28h in plasma, and it can pass through the blood-brain barrier, further indicating that such compounds can be used for in vivo research and treatment of diseases related to ferroptosis.
  • Example 8 Compounds can be used as fluorescent probes to detect ferroptosis
  • Fluorescence spectra of antioxidant fluorescent probe I-1 in different solvents In different solvents, the fluorescent probe was added with an initial concentration of 1 mM to make the concentration of the fluorescent probe in the solution 20 ⁇ M. Then, the fluorescence spectra in different solvents were tested with a fluorescence spectrometer. The excitation wavelength of the fluorescence spectrum was 350 nm and the emission wavelength was 525 nm. As shown in Figure 4, with the increase of solvent polarity, the maximum emission wavelength of I-1 gradually increased.
  • OS-RC2 cells were incubated with probe I-1, and the control group was incubated with antioxidant Fer-1.
  • the cells were cultured in an incubator at 37°C in a humid environment with 5% CO2 .
  • the above cells were then incubated with RSL3 for 1 hour, and the RSL3 group was set as a reference control.
  • the cells were incubated with BODIPY 581/591 for 30 minutes and then subjected to flow cytometry testing. Cells without any treatment were used as blank controls.
  • the cells treated with I-1 can effectively reverse the process of cell ferroptosis, which is basically consistent with the results of the Fer-1 control group.
  • the I-1 channel showed obvious fluorescence changes ( Figure 5b), indicating that the antioxidant fluorescent probe prepared by the present invention can effectively reverse the process of cell ferroptosis and has self-indication ability.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Diabetes (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Hematology (AREA)
  • Vascular Medicine (AREA)
  • Hospice & Palliative Care (AREA)
  • Emergency Medicine (AREA)
  • Endocrinology (AREA)
  • Psychiatry (AREA)
  • Obesity (AREA)
  • Materials Engineering (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Optics & Photonics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)

Abstract

本发明公开了一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物及其制备方法和应用。所述化合物的结构式为：本发明经过实验验证，所述化合物表现出强效的铁死亡抑制活性，良好的代谢稳定性，适用于铁死亡相关疾病的治疗；同时该类结构可作为特异性铁死亡探针使用，有氧化态和还原态两种形态，还原态时探针表现为绿色荧光，而氧化态时表现为蓝色荧光，其荧光变化可以作为该抗氧化剂作用程度的一种指示剂，又可以检测待测样品铁死亡进行情况，尤其是用于ONOO-高灵敏检测。

Description

一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物及其制备方法和应用 技术领域
本发明属于医药技术领域，具体涉及一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物及其制备方法和应用。
背景技术
细胞死亡大致可分为两大类：由过度细胞损伤引起的不受控制的细胞死亡以及依赖严格控制的分子通路的调控细胞死亡。凋亡是调节细胞死亡的最典型形式，通过激活caspases引发细胞死亡。铁死亡(ferroptosis)是近几年发现的一种新的细胞死亡方式，是在多种原因诱导下发生的氧化性细胞死亡，具有铁离子依赖性，其发生是细胞内脂质活性氧(reactive oxygen species,ROS)生成与降解的平衡失调所致。在发现铁死亡的最初几年中阐明的控制铁死亡机制主要围绕半胱氨酸和谷胱甘肽代谢，以及磷脂过氧化物酶GPX4防止过氧化脂质积累。铁死亡诱导剂通过不同的通路直接或间接作用于谷胱甘肽过氧化物酶(glutathione peroxidase,GPXs)，导致细胞抗氧化能力降低、ROS堆积、最终引起细胞氧化性死亡。脂质、铁和半胱氨酸代谢之间复杂的相互作用已成为这一细胞死亡途径的重要调节因素。最近，对铁死亡的调控已成为一种有吸引力的策略，用于干预人类疾病，包括癌症、神经退行性疾病和缺血性疾病。
铁死亡能被铁螯合剂、亲脂抗氧化剂和/或ferrostatin-1(fer-1)所抑制。Fer-1是一种具有抗氧化性能的芳基烷基胺，是最早被鉴定为铁死亡抑制剂之一。Fer-1作为脂质过氧化还原剂，通过氢原子转移或直接还原拦截和清除脂质自由基。然而其半衰期较短，不适合进一步的药理学评价。
近年来，高灵敏度、高分辨率的荧光检测技术一直受到研究人员的青睐，被广泛应用于各种物质的检测。与其它监测手段相比，荧光成像技术反应灵敏、准确，可实时监测且准确度较高，因此在铁死亡相关科研活动中，荧光探针也常被用于评价细胞铁死亡进程和铁死亡抑制剂的作用水平。过氧化亚硝酰阴离子(ONOO-)是一种重要的氧化物种，研究表明发现与铁死亡有着密不可分的联系，许多基于发色团的荧光探针已被应用于生物体内的ONOO-识别，如罗丹明和尼罗红化合物。但是这些小分子仅有识别功能，不适合作为抗氧化药物使用，因此在实际应用上面仍然具有一定的局限性。
通常情况下，荧光探针检测物质是依靠于荧光强度的增加或消减。基于此设计一种可以与ONOO-反应的荧光探针，此探针可以通过化学反应消耗ONOO-从而发挥抗氧化的作用，同时反应前后引起的荧光光谱变化使其具备实时监测功能。此类具有自指示功能的抗氧化剂荧光探针能够在逆转铁死亡过程中实现自我监测，不需要额外处理实验样本，这为开发铁死亡抗氧化剂的评价手段提供了一种全新的思路。
发明内容
本发明针对现有技术的不足，提供了一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物及其制备方法和应用。所述化合物抗氧化活性强，代谢稳定性好，适用于铁死亡相关疾病的治疗；且具有明显的荧光光谱变化，对ONOO-具有极强的选择性，现象明显且便于识别。
本发明的目的是通过如下技术方案实现的：
一方面，本发明提供了一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物，其结构式如下所示：
式中，R1选自氢、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C3-C8环烷氧基、氨基、苯基、苄基、萘基、C5-C10芳香性杂环基或C3-C7饱和杂环基；
R2选自C0～C8烷基、C3～C12环烷基、金刚烷基或多炔基；
R3选自氢、烷基、芳基、C1-C6烷基-芳基、C1-C6烷基-酚基或C3～C10环烷基；
其中，C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C3-C8环烷氧基、苯基、苄基、萘基、C5-C10芳香性杂环基、C3-C7饱和杂环基、C3～C12环烷基、多炔基、芳基、C1-C6烷基-芳基、C1-C6烷基-酚基或C3～C10环烷基能够被一个或多个原子或基团取代；
n为柔性烷基链的个数，n＝0或2-4。
优选地，所述R1选自C1-C6烷基、C1-C6烷氧基、C3-C8环烷基、C3-C8环烷氧基、芳基及衍生物。
优选地，所述R2选自环丙基、环丁基、环戊基、环己基及其衍生物、环庚基、金刚烷基及衍生物。
优选地，所述R3选自环丙基、环戊基、环己基、芳苄基及其衍生物。
优选地，n＝0、2-4。
优选地，所述化合物具体为化合物I-1、II-1～II-9、III-1～III-15、IIII-1～IIII-12，其结构式具体如下：


另一方面，本发明还提供了所述的含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物的制备方法，其包括以下步骤：
(1)将化合物4-氯-3-硝基苯甲酸、NH2NHBoc、HBTU和DIEPA加入到DMF中，室温反应4小时，反应结束后将混合液经分离提纯，得到淡黄色固体化合物2；
(2)将所述步骤(1)的化合物2和劳森试剂加入到二氧六环中，在110℃条件下反应过夜，反应结束后经分离提纯，得到噻二唑类化合物3；
(3)将所述步骤(2)的噻二唑类化合物3和相应的胺加入到DMSO中，60℃反应18小时，反应结束后经分离提纯，得到相应中间体化合物4；
(4)将所述步骤(3)的中间体化合物4和Pd/C加入到甲醇中，在H2条件下，室温搅拌6小时，反应至结束后经分离提纯，得到含氨基类化合物5；
(5)将所述步骤(4)的含氨基类化合物5、相应的醛和NaBH(OAc)3加入到DCM中，室温搅拌6小时，反应至结束后经分离提纯，得到含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物6。
优选地，所述步骤(1)中4-氯-3-硝基苯甲酸、NH2NHBoc、HBTU和DIEPA的摩尔比为1:1.5:1.5:1.5。
优选地，所述步骤(2)中化合物2和劳森试剂的摩尔比为1:2.5。
优选地，所述步骤(3)中噻二唑类化合物3和相应的胺的摩尔比为1:1.5。
优选地，所述步骤(4)中中间体化合物4和Pd/C的质量比为10:1。
优选地，所述步骤(5)中含氨基类化合物5、相应的醛和NaBH(OAc)3的摩尔比1:1.1:2。
优选地，所述步骤(1)中分离提纯的具体方法为：反应结束后将混合液倒入30mL水中，乙酸乙酯萃取水层，有机层经干燥、浓缩，混合物经柱层析分离得到淡黄色固体化合物2。
优选地，所述步骤(2)中分离提纯的具体方法为：反应结束后，冷却至室温，将溶剂蒸出，混合物用水和乙酸乙酯萃取，合并有机层，再用10％碳酸氢钠溶液洗涤合并的有机层。然后用硫酸钠干燥有机层，过滤，浓缩并经柱层析分离得到噻二唑类化合物3。
优选地，所述步骤(3)中分离提纯的具体方法为：反应结束后将混合反应液倒入30mL水中，乙酸乙酯萃取水层，有机层经干燥、浓缩，混合物经柱层析分离得到中间体化合物4。
优选地，所述步骤(4)中分离提纯的具体方法为：反应结束后用硅藻土过滤溶液，在真空下蒸出溶剂，粗品经柱层析分离得到含氨基类化合物4；
优选地，所述步骤(5)中分离提纯的具体方法为：反应结束后将反应液倒入饱和碳酸氢钠水溶液中水层用CH2Cl2萃取，用饱和碳酸氢钠水溶液洗涤合并的有机层，然后用盐水洗涤，干燥，过滤，真空浓缩，混合物经柱层析分离出得到含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物。
另一方面，本发明还提供了所述的含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物在制备铁死亡抑制剂中的应用。
另一方面，本发明还提供了所述的含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物在制备铁死亡相关疾病的药物中的应用。
进一步的，所述铁死亡相关疾病包括神经退行性、组织缺血再灌注损伤、脑卒中、心血管、肝肾衰竭、炎症、糖尿病并发症。
进一步的，所述药物中含有含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物或其药学上可接受的盐、赋形剂或载体。
另一方面，本发明还提供了所述的含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物在制备抗氧化荧光探针或抗氧化指示剂中的应用。
进一步的，所述抗氧化荧光探针或抗氧化指示剂用于ONOO-高灵敏检测，能够有效的逆转细胞铁死亡进程并且具备自指示能力。
本发明与现有技术相比，具有以下优点和有益效果：
1、本发明设计获得了一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物，其可有效抑制铁死亡，且代谢稳定性好，可用于铁死亡相关疾病研究；
2、本发明所述的含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物的制备方法，其制备方法简便、产率高，适合大规模推广应用；
3、本发明所述的含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物，可作为所述荧光探针，具有明显的荧光光谱变化，在抑制铁死亡过程中可以通过自身荧光光谱的变化实时评估监测作用效果。
附图说明
图1A为化合物I-1的核磁共振氢谱；图1B为化合物I-1的碳谱；图1C为化合物I-1的高分辨质谱；
图2为抗氧化荧光探针I-1检测ONOO-的选择性测试结果图，其中1-14分别代表生物活性小分子Blank，Cu2+，Zn2+，Fe3+，Fe2+，Al3+，Ca2+，SO4 2-，SO3 2-，NO2 -，O2-，t-BuOOH，H2O2，HClO，ONOO-，·OH，Vc-，GSH，VC；
图3为抗氧化荧光探针I-1检测ONOO-的荧光光谱结果图；
图4为抗氧化荧光探针I-1检测在不同溶剂中荧光光谱结果图；
图5为流式细胞术评估I-1抗氧化活性以及其自指示能力结果图。
具体实施方式
下面对本发明的实施例作详细说明，本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物的总合成路线如下：
其中，(a)HBTU,DIEPA,NH2NHCOR1；(b)劳氏试剂，回流；(c)相应的胺,K2CO3,DMSO,80℃；(d)Pd/C,H2,CH3OH；(e)相应的醛，NaBH(OAc)3,DCM。
(1)合成方法a：将化合物1(4.96mmol)，NH2NHCOR1(7.44mmol)，HBTU(7.44mmol)和DIEPA(7.44mmol)加入到装有DMF(3mL)的反应瓶中，室温反应至完全。将混合液倒入30mL水中，乙酸乙酯萃取水层，有机层经干燥、浓缩，混合物经柱层析分离得得到淡黄色固体2。
(2)合成方法b：将上一步得到的化合物2(0.78mmol)和劳森试剂(1.94mmol)加入到反应瓶中，二氧六环作溶剂，在110℃条件下反应过夜。反应结束后，冷却至室温，将溶剂蒸出，混合物用水和乙酸乙酯萃取，合并有机层，再用10％碳酸氢钠溶液洗涤合并的有机层。然后用硫酸钠干燥有机层，过滤，浓缩并经柱层析分离，得到黄色固体，为噻二唑类化合物3。
(3)合成方法c：将上一步得到的化合物3(0.45mmol)和相应的胺(0.67mmol)加入反应瓶中，DMSO(3mL)作溶剂，反应液在60℃反应至TLC检测反应结束。将混合反应液倒入30mL水中，乙酸乙酯萃取水层，有机层经干燥、浓缩，混合物经柱层析分离得到黄色固体4。
(4)合成方法d：将化合物4(0.35mmol)溶于甲醇中，并在室温下用10％Pd/C在H2条件下反应6小时。用硅藻土垫过滤溶液，并在减压下除去溶剂。残留物通过硅胶快速柱色谱纯化，得到所需化合物5。
(5)合成方法e：将化合物5(0.2mmol)加入到含有苯甲醛(0.23mmol)的DCM溶液中，并在室温下搅拌30分钟。随后一次性加入三乙酰氧基硼氢化钠(0.4mmol)，将反应物搅拌24小时。然后倒入饱和碳酸氢钠水溶液中水层用CH2Cl2萃取。用饱和碳酸氢钠水溶液洗涤合并的有机层，然后用盐水洗涤，干燥，过滤，真空浓缩，混合物经柱层析分离出得到目标化合物6。
实施例1：化合物I-1的制备
本实施例采用上述描述的实验方法制得化合物I-1。1H NMR(400MHz,Chloroform-d)δ7.39(s,1H),7.29(d,J＝7.1Hz,1H),6.62(d,J＝8.3Hz,1H),3.29(m,1H),2.74(s,3H),2.09–2.04(m,2H),1.80–1.64(m,4H),1.46–1.32(m,4H).13C NMR(101MHz,Chloroform-d)δ169.61,163.04,139.85,133.40,122.06,119.39,115.92,111.11,51.66,33.27,29.72,25.87,24.94,15.72.HRMS(ESI)for C15H20N4S[M+H]+calcd 289.1487,found 289.1481(图1).
实施例2：化合物II-1～II-9的制备
本实施例采用上述描述的实验方法制得化合物II-1～II-9：
1H NMR(400MHz,Chloroform-d)δ7.41(d,J＝1.9Hz,1H),7.34(dd,J＝8.2,1.9Hz,1H),6.63(d,J＝8.2Hz,1H),2.92(s,3H),2.76(s,3H).13C NMR(101MHz,DMSO-d6)δ169.82,162.79,140.46,135.41,118.98,118.18,112.14,108.87,30.30,15.65.
1H NMR(400MHz,Chloroform-d)δ7.41(d,J＝1.9Hz,1H),7.31(dd,J＝8.2,1.9Hz,1H),6.63(d,J＝8.2Hz,1H),3.24–3.19(m,2H),2.76(s,3H),1.31(t,J＝7.2Hz,3H).13C NMR(101MHz,Chloroform-d)δ169.66,163.04,141.18,133.40,122.04,119.65,115.27,110.37,38.37,22.70,15.71.
1H NMR(400MHz,Chloroform-d)δ7.41(d,J＝2.0Hz,1H),7.31(dd,J＝8.3,2.0Hz,1H),6.64(d,J＝8.3Hz,1H),3.72–3.65(m,1H),2.76(s,3H),1.27(d,J＝6.3Hz,6H).13C NMR(101MHz,Chloroform-d)δ169.66,163.03,141.37,133.29,122.15,115.52,110.36,51.60,28.03,22.68,20.59,15.72.
1H NMR(400MHz,Chloroform-d)δ7.42(d,J＝1.9Hz,1H),7.31(dd,J＝8.2,1.7Hz,1H),6.62(d,J＝8.3Hz,1H),2.99(d,J＝6.8Hz,2H),2.76(s,3H),1.95(m,1H),1.03(d,J＝6.7Hz,6H).13C NMR(101MHz,Chloroform-d)δ168.65,161.99,140.41,132.29,121.10,118.38,114.46,109.26,50.55,27.01,19.57,14.69.
1H NMR(400MHz,Chloroform-d)δ7.40(s,1H),7.30(d,J＝7.8Hz,1H),6.71–6.67(m,1H),3.88–3.81(m,1H),2.76(s,3H),2.07(m,2H),1.80–1.73(m,2H),1.69–1.64(m,2H),1.59–1.55(m,2H).13C NMR(101MHz,Chloroform-d)δ169.66,163.00,140.81,133.49,122.01,119.42,115.36,119.42,115.36,111.25,54.42,33.63,29.71,24.26,15.71.HRMS(ESI)for C14H18N4S[M+H]+calcd 275.1330,found 275.1325.
1H NMR(400MHz,Chloroform-d)δ7.40(s,1H),7.30(d,J＝8.1Hz,1H),6.56(d,J＝8.3Hz,1H),3.50(s,1H),2.76(s,3H),2.02(m,2H),1.62(m,10H).13C NMR(101MHz,Chloroform-d)δ169.68,162.96,139.95,133.49,122.02,119.15,115.66,111.00,53.52,34.70,28.29,24.42,15.70.HRMS(ESI)for C16H22N4S[M+H]+calcd 303.1643,found 303.1638.
1H NMR(400MHz,Chloroform-d)δ7.39(d,J＝2.0Hz,1H),7.22(dd,J＝8.3,2.1Hz,1H),6.98(d,J＝8.3Hz,1H),2.77(s,3H),2.14(s,3H),1.96(m,6H),1.68(m,6H).13C NMR(101MHz,Chloroform-d)δ169.23,163.48,141.96,136.13,132.28,125.65,120.86,115.92,42.48,36.32,29.64,22.70,15.74.
1H NMR(400MHz,Chloroform-d)δ7.38(d,J＝1.6Hz,1H),7.29(s,1H),6.59(d,J＝8.2Hz,1H),5.00(s,1H),3.50–3.42(m,2H),3.30(t,J＝5.5Hz,2H),2.76(s,3H),1.45(s,9H).13C NMR(101MHz,Chloroform-d)δ169.73,163.12,156.95,140.45,133.68,121.58,119.63,114.84,109.95,44.77,39.94,29.69,28.38,15.68.
1H NMR(400MHz,Chloroform-d)δ7.42(d,J＝2.0Hz,1H),7.31(dd,J＝8.2,2.0Hz,1H),6.65(d,J＝8.3Hz,1H),4.15–3.97(m,2H),3.50(m,1H),2.97(t,J＝11.8Hz,2H),2.76(s,3H),2.10–2.03(m,2H),1.47(s,9H).13C NMR(101MHz,Chloroform-d)δ169.42,163.24,154.77,139.09,133.77,121.90,120.18,116.13,111.48,79.74,50.02,32.13,29.70,28.44,15.72.HRMS(ESI)for C19H27N5O2S[M+H]+calcd 390.1964,found 390.1958.
实施例3：化合物III-1～III-15的制备
本实施例采用上述描述的实验方法制得化合物III-1～III-15：
1H NMR(400MHz,Chloroform-d)δ7.44–7.35(m,5H),7.33–7.28(m,2H),6.66(d,J＝8.2Hz,1H),4.33(s,2H),3.35–3.26(m,1H),2.75(s,3H),2.10–2.02(m,2H),1.80–1.73(m,2H),1.72– 1.61(m,2H),1.45–1.33(m,4H).13C NMR(101MHz,Chloroform-d)δ170.00,162.90,139.80,139.00,136.22,128.68,128.15,127.48,121.13,111.61,110.60,51.60,49.20,33.38,29.70,25.89,24.99.HRMS(ESI)for C22H26N4S[M+H]+calcd 379.1956,found 379.1951.
1H NMR(400MHz,Chloroform-d)δ8.57(d,J＝5.8Hz,2H),7.33(d,J＝5.7Hz,2H),7.29(dd,J＝6.1,1.8Hz,2H),6.69(d,J＝8.8Hz,1H),4.40(s,2H),3.32(m,1H),2.73(s,3H),2.13–2.03(m,2H),1.73(m,4H),1.48–1.26(m,4H).13C NMR(101MHz,Chloroform-d)δ169.73,163.06,150.06,148.19,139.86,135.54,122.61,121.62,119.90,111.77,111.18,51.64,47.75,33.40,25.88,24.94,15.71.HRMS(ESI)for C22H25N5S[M+H]+calcd 380.1909,found 380.1903.
1H NMR(400MHz,Chloroform-d)δ8.76(d,J＝5.7Hz,2H),8.66(s,1H),7.79(d,J＝2.0Hz,1H),7.76–7.73(m,2H),7.58(dd,J＝8.5,2.0Hz,1H),6.69(d,J＝8.6Hz,1H),3.45–3.35(m,1H),2.77(s,3H),2.13–2.05(m,2H),1.80(m,2H),1.67(m,2H),1.48–1.31(m,4H).13C NMR(101MHz,Chloroform-d)δ169.26,162.94,155.25,150.51,145.53,142.84,134.78,129.66,122.23,117.63,115.54,110.52,51.17,33.09,25.76,24.82,15.74.
1H NMR(400MHz,Chloroform-d)δ7.38(d,J＝1.9Hz,1H),7.21(m,3H),6.91(d,J＝8.4Hz,2H),6.63(d,J＝8.3Hz,1H),4.02(s,2H),3.28(m,1H),2.75(s,3H),2.04(t,J＝4.7Hz,2H),1.80–1.72(m,2H),1.68–1.61(m,1H),1.38(dd,J＝16.5,8.3Hz,5H).13C NMR(101MHz, Methanol-d4)δ171.45,156.17,139.45,135.92,130.14,128.69,128.41,124.13,119.28,117.62,114.81,114.66,110.02,109.49,63.70,51.46,32.78,30.42,29.31,25.69,24.90.
1H NMR(400MHz,Chloroform-d)δ7.38(td,J＝5.6,3.0Hz,3H),7.29(dd,J＝8.2,2.0Hz,1H),7.07–7.02(m,2H),6.66(d,J＝8.3Hz,1H),4.30(s,2H),3.31(m,1H),2.74(s,3H),2.10–2.03(m,2H),1.77(m,2H),1.69–1.58(m,2H),1.45–1.30(m,4H).13C NMR(101MHz,Chloroform-d)δ169.92,162.97,139.77,136.02,129.61,121.32,115.62,115.41,111.73,110.81,51.65,48.44,33.38,29.70,25.88,24.97,15.71.HRMS(ESI)for C22H25FN4S[M+H]+calcd 397.1862,found 397.1857.
1H NMR(400MHz,Chloroform-d)δ7.40(d,J＝1.9Hz,1H),7.33–7.28(m,3H),7.18(d,J＝7.9Hz,2H),6.65(d,J＝8.3Hz,1H),4.28(s,2H),3.29(m,1H),2.74(s,3H),2.36(s,3H),2.05(m,2H),1.77(dm,2H),1.67(m,1H),1.43–1.36(m,2H),1.23–1.14(m,3H).13C NMR(101MHz,Chloroform-d)δ170.03,162.88,139.70,137.16,136.30,135.94,129.35,128.18,121.05,111.52,110.57,51.62,48.94,33.36,25.89,25.00,21.14,15.69.
1H NMR(400MHz,Chloroform-d)δ8.27(s,1H),8.16–8.09(m,1H),7.72(d,J＝7.6Hz,1H),7.51(t,J＝7.9Hz,1H),7.31(d,J＝1.9Hz,1H),7.27(m,1H),6.68(d,J＝8.3Hz,1H),4.46(s,2H),3.31(m,1H),2.73(s,3H),2.12–2.04(m,2H),1.78(m,2H),1.72–1.59(m,2H),1.45–1.25(m,4H). 13C NMR(101MHz,Chloroform-d)δ169.85,163.00,148.51,141.31,139.97,135.44,133.98,129.54,122.61,122.43,121.68,119.62,111.57,111.02,51.69,48.13,33.33,25.89,24.98,15.69.
1H NMR(400MHz,Chloroform-d)δ7.40(d,J＝1.9Hz,1H),7.31(m,3H),7.15–7.12(m,1H),6.66(d,J＝8.2Hz,1H),4.35(s,2H),3.30(m,1H),2.75(s,3H),2.10–2.03(m,2H),1.81–1.74(m,2H),1.71–1.51(m,3H),1.45–1.29(m,3H).13C NMR(101MHz,Chloroform-d)δ169.93,162.98,145.52,139.94,129.26,127.58,126.18,122.28,121.61,111.89,44.37,33.26,29.70,25.87,24.97,15.72.HRMS(ESI)for C20H24N4S2[M+H]+calcd 385.1521,found 385.1515.
1H NMR(400MHz,Chloroform-d)δ8.14(d,J＝8.5Hz,1H),8.09(d,J＝8.6Hz,1H),7.82(d,J＝8.4Hz,1H),7.77–7.70(m,1H),7.53(t,J＝7.9Hz,1H),7.46–7.41(m,2H),7.29–7.26(m,1H),6.69(d,J＝8.1Hz,1H),4.66(s,2H),3.38(m1H),2.74(s,3H),2.13(dd,J＝12.5,3.7Hz,2H),1.82(m,2H),1.71–1.63(m,1H),1.47–1.31(m,5H).13C NMR(101MHz,Chloroform-d)δ169.65,164.33,151.96,150.30,147.20,143.51,136.79,129.98,127.76,127.58,122.56,122.25,120.83,114.03,57.55,51.58,31.49,29.71,26.31,15.78.
1H NMR(400MHz,Chloroform-d)δ7.36(s,1H),7.23(s,1H),6.65(d,J＝8.2Hz,1H),3.80(p,J＝6.2Hz,1H),3.31–3.19(m,1H),2.75(s,3H),2.38–2.30(m,2H),2.05(m,4H),1.75(m,5H),1.67–1.58(m,5H),1.52(m,2H).13C NMR(101MHz,Chloroform-d)δ160.76,144.91,122.30,121.81,119.12,111.34,108.52,49.39,42.83,29.27,28.02,27.49,23.76,22.79,21.00,13.52.
1H NMR(400MHz,Chloroform-d)δ7.35(s,2H),6.63(s,1H),3.84(dd,J＝14.3,7.9Hz,1H),3.36–3.18(m,1H),2.74(s,3H),2.08(dt,J＝12.3,6.2Hz,4H),1.82–1.71(m,4H),1.70–1.59(m,4H),1.57–1.45(m,3H),1.44–1.33(m,3H).13C NMR(101MHz,Chloroform-d)δ162.87,136.05,120.71,119.71,112.45,110.72,108.28,55.07,51.63,33.59,33.43,29.70,25.93,24.98,24.32,15.70.
1H NMR(400MHz,Chloroform-d)δ7.47–7.26(m,2H),6.62(d,J＝7.1Hz,1H),3.67–3.53(m,1H),3.36–3.16(m,1H),2.74(s,3H),2.06(d,J＝10.8Hz,2H),1.82–1.73(m,2H),1.66(dt,J＝12.0,3.2Hz,1H),1.42–1.24(m,11H).13C NMR(101MHz,Chloroform-d)δ165.54,163.03,141.19,124.47,123.98,5119.11,112.99,111.42,54.53,52.52,41.99,35.69,33.54,31.94,31.44,29.37,25.99,25.00,24.29,22.70,14.13.
1H NMR(400MHz,Chloroform-d)δ7.34(s,1H),7.22(d,J＝6.8Hz,1H),6.63(d,J＝8.1Hz,1H),3.53(d,J＝5.1Hz,1H),3.27(s,1H),2.73(s,3H),2.06(t,J＝14.2Hz,3H),1.75(d,J＝12.3Hz,4H),1.65(m,3H),1.54(m,2H),1.42–1.26(m,7H),0.91(dd,J＝11.8,6.4Hz,3H).13C NMR(101MHz,Chloroform-d)δ170.10,162.89,140.65,135.45,120.89,119.80,113.31,111.46,65.23,51.54,41.98,34.00,33.36,32.42,29.95,25.93,24.84,22.25,15.67.
1H NMR(400MHz,Chloroform-d)δ7.36(s,1H),7.25–7.19(m,1H),6.62(d,J＝7.3Hz,1H),3.35–3.18(m,2H),2.74(s,3H),2.09–2.01(m,4H),1.82–1.72(m,4H),1.69–1.63(m,2H),1.37(m,5H),1.28(m,5H).13C NMR(101MHz,Chloroform-d)δ162.88,140.81,135.06,121.23,113.60,110.96,52.52,51.53,41.98,33.60,29.66,26.02,24.99,15.68.
1H NMR(400MHz,Chloroform-d)δ7.09(dd,J＝8.0,1.7Hz,1H),7.04(d,J＝1.6Hz,1H),6.35(d,J＝8.0Hz,1H),3.15(m,1H),3.04–2.77(m,3H),2.71(s,3H),2.42–2.27(m,1H),2.09(m,3H),1.80(dd,J＝29.5,12.8Hz,4H),1.70–1.59(m,3H),1.37–1.26(m,3H),1.10(d,J＝5.9Hz,6H).13C NMR(101MHz,Chloroform-d)δ170.08,162.28,142.10,138.80,122.12,117.76,104.78,52.85,45.46,30.25,29.70,26.21,25.52,15.68.
实施例4：化合物IIII-1～IIII-11的制备
本实施例采用上述描述的实验方法制得化合物IIII-1～IIII-11：
1H NMR(400MHz,Chloroform-d)δ7.39(d,J＝2.0Hz,1H),7.29(dd,J＝8.3,2.0Hz,1H),6.62(d,J＝8.4Hz,1H),3.30(m,1H),3.09(q,J＝7.6Hz,2H),2.10–2.03(m,2H),1.95–1.87(m,1H),1.77(m,2H),1.69–1.57(m,2H),1.45–1.34(m,6H).13C NMR(101MHz,Chloroform-d)δ169.91,160.41,139.67,133.49,121.93,119.49,115.89,111.17,51.71,33.22,25.86,24.94,23.85,14.40.
1H NMR(400MHz,Chloroform-d)δ7.41(d,J＝2.0Hz,1H),7.30(dd,J＝8.2,2.0Hz,1H),6.63(d,J＝8.3Hz,1H),3.31(m,1H),3.08–3.02(m,2H),2.12–2.03(m,2H),1.89–1.76(m,4H),1.44–1.32(m,4H),1.21(m,2H),1.04(t,J＝7.4Hz,3H).13C NMR(400MHz,CDCl3)δ169.14,168.40,139.76,133.45,122.01,119.55,115.93,111.17,51.68,33.26,32.11,25.87,24.95,23.47,13.64.
1H NMR(400MHz,Chloroform-d)δ7.37(d,J＝2.0Hz,1H),7.27(d,J＝2.1Hz,1H),7.25(s,1H),6.63(d,J＝8.3Hz,1H),3.31(m,1H),2.38(m,1H),2.08(m,2H),1.83–1.75(m,2H),1.67(dm,1H),1.47–1.28(m,3H),1.24–1.16(m,4H),1.14–1.08(m,2H).13C NMR(400MHz,CDCl3)δ171.56,167.58,133.48,121.84,115.83,111.24,51.70,33.23,29.70,25.86,24.94,11.67,11.20.
1H NMR(400MHz,Chloroform-d)δ7.44(d,J＝2.1Hz,1H),7.37(dd,J＝8.3,2.1Hz,1H),6.64(d,J＝8.4Hz,1H),3.34(m,1H),2.12–2.03(m,2H),1.79(dt,J＝13.2,3.6Hz,2H),1.67(m,1H),1.46–1.34(m,2H),1.30–1.24(m,3H).13C NMR(101MHz,Chloroform-d)δ172.62,154.36,133.35,123.02,116.39,111.10,51.76,33.12,29.71,25.77,24.89.
1H NMR(400MHz,Chloroform-d)δ8.00–7.96(m,2H),7.50–7.45(m,4H),7.40(dd,J＝8.3,2.1Hz,1H),6.66(d,J＝8.4Hz,1H),3.33(m,1H),2.14–2.04(m,2H),1.80(m,2H),1.68(m,2H),1.44–1.35(m,2H),1.30(q,J＝3.2Hz,2H).13C NMR(400MHz,CDCl3)δ168.80,166.40,133.41,130.68,130.54,129.09,127.76,121.20,116.26,113.06,50.85,33.09,29.71,25.82,24.93.
11H NMR(400MHz,Chloroform-d)δ7.83(d,J＝8.6Hz,2H),7.59(d,J＝8.6Hz,2H),7.46(d,J＝1.9Hz,1H),7.38(dd,J＝8.3,1.9Hz,1H),6.65(d,J＝8.4Hz,1H),3.33(m,1H),2.13–2.03(m,2H),1.79(m,2H),1.68(m,2H),1.46–1.33(m,2H),1.31–1.22(m,4H).13C NMR(101MHz,Chloroform-d)δ165.17,132.29,129.06,124.99,122.28,116.13,51.89,33.12,29.71,25.82,24.92.
1H NMR(400MHz,Chloroform-d)δ7.37(d,J＝2.1Hz,1H),7.36–7.31(m,4H),7.29(dd,J＝6.5,2.2Hz,1H),7.25(d,J＝5.9Hz,1H),6.60(d,J＝8.3Hz,1H),4.41(s,2H),3.30(m,1H),2.10–2.02(m,2H),1.78(m,2H),1.67(m,1H),1.46–1.36(m,2H),1.29(d,J＝3.1Hz,1H),1.21(t,J＝3.2Hz,2H).13C NMR(400MHz,CDCl3)δ170.10,167.75,140.05,137.48,133.28,128.94,128.86,127.36,122.07,119.16,115.90,110.89,51.55,36.56,33.26,25.85,24.92.
1H NMR(400MHz,Chloroform-d)δ9.12(s,1H),8.68(d,J＝3.6Hz,1H),8.33(dt,J＝8.0,1.8Hz,1H),7.48(d,J＝2.0Hz,1H),7.42(dt,J＝9.6,5.1Hz,2H),6.66(d,J＝8.3Hz,1H),3.39–3.27(m,1H),2.12–2.05(m,2H),1.79(m,2H),1.67(m,1H),1.45–1.35(m,2H),1.28(m,3H).13C NMR(101MHz,Chloroform-d)δ180.18,151.32,148.62,134.59,123.93,116.27,52.22,33.08,29.70,25.77,24.91.
1H NMR(400MHz,Chloroform-d)δ7.52–7.23(m,8H),6.68(d,J＝8.0Hz,1H),4.32(s,2H),3.34(t,J＝10.0Hz,1H),2.08(d,J＝11.1Hz,2H),1.79(d,J＝13.1Hz,2H),1.68(d,J＝12.6Hz,1H),1.40(m,2H),1.28–1.20(m,3H).13C NMR(101MHz,Chloroform-d)δ173.10,154.99,154.61,154.22,153.83,141.37,138.68,135.96,128.75,128.12,127.61,122.22,117.47,112.07,110.25,51.58,49.18,33.29,25.82,24.96.
1H NMR(400MHz,Chloroform-d)δ7.40(s,1H),7.33(d,J＝8.2Hz,1H),6.63(d,J＝8.2Hz,1H),3.36–3.27(m,1H),3.22(t,J＝10.0Hz,1H),2.11–2.01(m,4H),1.82–1.73(m,4H),1.67(d,J＝11.0Hz,2H),1.45–1.34(m,4H),1.26(d,J＝11.3Hz,6H).13C NMR(101MHz,Chloroform-d)δ173.16,154.96,154.58,154.19,153.80,142.34,134.80,122.30,117.27,114.10,110.48,52.58,51.45,33.60,33.25,29.72,25.99,25.87,24.97,24.89.
1H NMR(400MHz,Chloroform-d)δ7.38(s,1H),7.34(d,J＝8.1Hz,1H),6.63(d,J＝8.2Hz,1H),3.87–3.79(m,1H),3.36–3.26(m,1H),2.13–2.01(m,4H),1.77(m,4H),1.70–1.60(m,3H),1.52(m,2H),1.44–1.34(m,2H),1.29(s,3H).13C NMR(101MHz,Chloroform-d)δ172.25,153.95,153.56,153.18,152.79,140.63,134.87,120.74,116.48,111.88,109.20,54.08,50.54,32.58,32.29,24.86,23.95,23.35.
实施例5：化合物IIII-12的制备
本实施例采用下述描述的实验方法制得化合物IIII-12：
其中，(a)NaBH3CN,CH3COOH；(B)CbzCl,TEA,DCM；(c)CF3COOH,NaNO2；(d)LiOH,THF；(e)HBTU,DIEPA,NH2NH2Ac；(f)Lawesson's Reagent,THF,reflux；(g)H2,Pd/C,CH3OH。
(1)1,2,3,4-四氢喹啉-6-甲酸甲酯(化合物2)的合成
将喹啉-6-甲酸甲酯(0.3g,1.6mmol)加入反应瓶中，冰乙酸(20mL)作溶剂，冰浴条件下分批加入NaBH3CN(0.302g,0.48mmol)，之后室温反应至结束，将反应液倾入饱和的NaHCO3水溶液，乙酸乙酯萃取，合并有机层并干燥，真空浓缩混合液，之后经柱层析分离得到白色固体(0.16g，52.28％)。
1H NMR(400MHz,DMSO-d6)δ7.48–7.43(m,2H),6.61(s,1H),6.42(d,J＝8.2Hz,1H),3.71(s,3H),3.22(m,2H),2.67(t,J＝6.2Hz,2H),1.80–1.73(m,2H).
(2)3-二氢喹啉-1,6(2H)-二羧酸1-苄基-6-甲基(化合物3)的合成
将1,2,3,4-四氢喹啉-6-甲酸甲酯(0.25g,1.3mmol)，DIEPA(0.67g,5.2mmol)溶于装有20mL二氯甲烷的反应瓶中，冰浴条件下逐滴加入Cbz-Cl(0.67g,3.9mmol)，之后室温反应至结束。将混合物经柱层析分离得到目标化合物(0.39g,91.7％)为白色固体。
1H NMR(400MHz,DMSO-d6)δ7.86(d,J＝8.4Hz,1H),7.71(d,J＝8.3Hz,2H),7.44–7.31(m,5H),5.21(s,2H),3.81(s,3H),3.79–3.73(m,2H),2.78(t,J＝6.4Hz,2H),1.86(p,J＝6.3Hz,2H).
(3)8-硝基-3,4-二氢喹啉-1,6(2H)-二羧酸1-苄基-6-甲基(化合物4)的合成
将3-二氢喹啉-1,6(2H)-二羧酸1-苄基-6-甲基(0.3g,0.92mmol)溶于装有三氟乙酸(5mL)的反应瓶中，冰浴条件下分批加入NaNO2(0.07g,1.01mmol)，冰浴条件下反应1h，之后室温反应2h。反应结束后将混合液倾入氨水水溶液中，EA萃取水层，合并有机层并干燥，真空浓缩混合液，之后经柱层析分离得到黄色油状物(0.21g，61.76％)。
1H NMR(400MHz,DMSO-d6)δ8.21(d,J＝1.9Hz,1H),8.07(d,J＝2.0Hz,1H),7.47–7.19(m,5H),5.31–5.02(m,2H),3.88(s,5H),3.05–2.72(m,2H),2.11–1.65(m,2H).
(4)6-(5-甲基-1,3,4-噻二唑-2-基)-8-硝基-3,4-二氢喹啉-1(2H)-羧酸苄酯(化合物7)的合成
将6-(2-乙酰肼-1-羰基)-8-硝基-3,4-二氢喹啉-1(2H)-羧酸苄酯(0.5g,1.21mmol)和劳森试剂(2.93g,7.26mmol)加入反应瓶中，二氧六环作溶剂(50mL)，回流反应过夜。将溶剂在真空条件下蒸出，剩余的混合物用乙酸乙酯溶解，碳酸氢钠饱和溶液洗涤有机层，之后合并有机层并浓缩，混合物经柱层析分离得到黄色油状物(0.25g,50.3％)。
1H NMR(400MHz,DMSO-d6)δ8.23(s,1H),8.06(s,1H),7.39–7.25(m,5H),5.16(s,2H),3.99–3.66(m,2H),2.89(s,2H),2.76(s,3H),2.00–1.81(m,2H).
(5)化合物IIII-12的合成
将化合物7(0.2g,0.48mmol)加入含有20mL甲醇溶液的反应瓶中，加入10％的钯碳(0.04g)，氢气条件下反应12h。反应结束后，用硅藻土过滤掉钯碳，混合液浓缩后经柱层析得到化合物IIII-12(0.07g,58.33％)。
1H NMR(400MHz,Chloroform-d)δ7.38(s,1H),7.25(s,1H),3.38–3.28(m,2H),2.83–2.73(m,5H),2.01–1.86(m,2H).13C NMR(101MHz,Chloroform-d)δ163.88,137.49,131.06,120.65,118.73,115.93,112.39,40.38,27.72,21.15,14.37.
实施例6：化合物的铁死亡抑制活性测试
研究显示，GPX4抑制剂如RSL-3能够诱导细胞发生铁死亡，这种铁死亡也可以被其他小分子物质阻断，如亲脂性抗氧化剂，如Ferrostatin-1(fer-1)、Liproxstatin等。因此，铁死亡抑制剂阻断铁死亡的能力可以通过对铁死亡诱导剂诱发的细胞铁死亡的逆转情况进行指示。
细胞株：人肾癌细胞株OS-RC-2、人神经母细胞瘤细胞SH-SY5Y，购自中国科学院上海细胞库。
方法：MTT法，具体如下：取对数生长期的人肾癌细胞株OS-RC-2/人神经母细胞瘤细胞SH-SY5Y，消化收集并稀释，以每孔约为4000-5000个细胞种于96孔板，实验设3个复孔，每孔80μL。置于37℃、5％CO2培养箱中培养过夜。实验分别设DMSO对照组和九个不同浓度的化合物给药组。给药组加入不同浓度的化合物，并设置DMSO对照组(与最高浓度化合物的稀释倍数相同)，置5％CO2、37℃的培养箱中继续孵育1h后，每个化合物浓度均加入浓度为5μM RSL3诱导铁死亡，并设置RSL3对照组和DMSO对照组，置5％CO2、37℃的培养箱中继续孵育48h。每孔加入5mg/mL的20μL MTT溶液，继续于37℃培养箱中培养OS-RC-2细胞2h，每孔加入100μL DMSO，平稳振荡10min，混匀。置于酶联免疫法检测仪上，在570nm波长下检测每孔光密度值(OD值)，实验重复3次。按照下列公式计算：存活率％＝实验组OD值/DMSO对照组OD值×100％。
结果参见表1，说明本发明的多种化合物能够明显抑制铁死亡，具有良好的铁死亡抑制活性。
表1：化合物的铁死亡抑制活性测试结果



表中：“A”指示IC50≤100nM，“B”指示IC50>100nM且≤500nM，“C”指示IC50>500
nM且≤1μM，“D”指示IC50>1μM且≤10μM。
实施例7：化合物的体外/内药物代谢性质
体外测试方法：将血浆、肝微粒体匀浆样品储存在-20℃，使用前取出，逐渐升至室温。将化合物溶解在乙腈中，制成4毫克/毫升储备溶液，然后加入血浆或肝微粒体匀浆，最终浓度为0.4毫克/毫升，并在37℃下培养所需的时间(0，0.25，0.5，1，2，4，8，12，24和24h)。在孵育的终止点终止乙腈，将混合物涡旋30s并在12000转/分钟离心10min，然后通过0.22μM过滤器过滤。滤液用HPLC系统(Agilent 1260 HPLC)进行分析，色谱柱为Alltima C18(5μm，4.6mm×250mm)。流速保持在400μL/min，初始流动条件为80％溶剂A(含0.1％乙酸的水)和20％溶剂B(含0.1％乙酸的甲醇)。溶剂B在0.50min内升高至80％，保持1.50min，接下来至5.00min，溶剂B升至100％，并在此保持3min。接下来在0.50min内，溶剂B降至初始条件(20％)，总运行时间为12.00min。在每个时间点剩余的化合物百分比＝化合物峰积分与内标峰积分的比值，内标峰面积为t＝0时间点的峰面积。
结果如下表2所示，本发明基于Fer-1结构的含噻二唑类化合物体外血浆和肝微粒体中表现出良好的稳定性，具有很好的ADME性质，可用于体内生物活性研究。
表2：ADME性质检测结果
体内测试方法：化合物在雄性小鼠体内单剂量静脉注射给药后，于0、0.5h、2h、4h、8h采集血样和脑组织，LC-MS/MS测定小鼠血浆、脑组织中化合物的浓度并计算相关药代参数，考察化合物在小鼠体内药代特征和脑组织分布情况。从结果可以看出，化合物I-1在小鼠体内表现出较好的代谢稳定性，在血浆中稳定性为半衰期2.28h，且其可以透过血脑屏障，进一步表明此类化合物可用于体内研究，治疗和铁死亡相关的疾病。
表3：I-1的药代参数
实施例8：化合物可作为荧光探针检测铁死亡
抗氧化荧光探针I-1检测ONOO-的选择性测试：在同样测试条件下，向溶液中加入过量的其它生物活性小分子，并测试荧光光谱，激发波长为350nm，最大发射波长分别为436nm和525nm。结果如图2所示，从1-14分别代表生物活性小分子Blank，Cu2+，Zn2+，Fe3+，Fe2+，Al3+，Ca2+，SO4 2-，SO3 2-，NO2 -，O2-，t-BuOOH，H2O2，HClO，ONOO-，·OH，Vc-，GSH，VC。F436/F525只有ONOO-存在时明显增强，其它生物活性小分子不对检测结果产生干扰，说明本发明制备的荧光探针对ONOO-具有较高的选择性。
抗氧化荧光探针I-1检测ONOO-的荧光光谱：在PBS缓冲液(pH＝7.4，0.5％DMSO)中，加入初始浓度为1mM的荧光探针，使溶液中荧光探针的浓度为10μM。然后，依次加入不同量的初始浓度为10mM的ONOO-，不加入ONOO-作为对照，37℃90min使ONOO-与荧光探针充分反应。用荧光光谱仪测试不同浓度ONOO-条件下的荧光光谱，荧光光谱的激发波长为350nm，发射波长为436nm、525nm。如图3所示，随着ONOO-浓度的增大，436nm处荧光强度逐渐增强，525nm处荧光强度逐渐减弱。
抗氧化荧光探针I-1在不同溶剂中荧光光谱：在不同溶剂中，加入初始浓度为1mM的荧光探针，使溶液中荧光探针的浓度为20μM。然后，用荧光光谱仪测试不同溶剂中的荧光光谱，荧光光谱的激发波长为350nm，发射波长为525nm，如图4所示，随着溶剂极性的增大，I-1的最大发射波长逐渐增大。
流式细胞术评估I-1抗氧化活性以及其自指示能力：OS-RC2细胞孵育探针I-1，对照组孵育抗氧化剂Fer-1，细胞在37℃的培养箱中，在5％的CO2下潮湿环境中培养。然后用RSL3分别孵育上述细胞1h，并设置RSL3组作为参比对照。最后用BODIPY581/591孵育细胞30min后进行流式细胞术测试，未经任何处理的细胞作为空白对照。由图5a可知，与RSL3组细胞相比，I-1处理过的细胞能够有效逆转细胞铁死亡进程，与Fer-1对照组结果基本一致，此外，I-1通道表现出明显的荧光变化(图5b)，说明本发明制备的抗氧化剂荧光探针能够有效的逆转细胞铁死亡进程并且具备自指示能力。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。

Claims (10)

  1. 一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物，其特征在于，所述化合物的结构式如下所示：
    式中，R1选自氢、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C3-C8环烷氧基、氨基、苯基、苄基、萘基、C5-C10芳香性杂环基或C3-C7饱和杂环基；
    R2选自C0～C8烷基、C3～C12环烷基、金刚烷基或多炔基；
    R3选自氢、烷基、芳基、C1-C6烷基-芳基、C1-C6烷基-酚基或C3～C10环烷基；
    其中，C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C3-C8环烷氧基、苯基、苄基、萘基、C5-C10芳香性杂环基、C3-C7饱和杂环基、C3～C12环烷基、多炔基、芳基、C1-C6烷基-芳基、C1-C6烷基-酚基或C3～C10环烷基能够被一个或多个原子或基团取代；
    n＝0或2-4。
  2. 根据权利要求1所述的化合物，其特征在于，所述化合物具体为化合物I-1、II-1～II-9、III-1～III-15、IIII-1～IIII-12，其结构式具体如下：

  3. 权利要求1所述的含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物的制备方法，其特征在于，所述制备方法包括以下步骤：
    (1)将4-氯-3-硝基苯甲酸、NH2NHBoc、HBTU和DIEPA加入到DMF中反应，结束后将混合液经分离提纯，得到淡黄色固体化合物；
    (2)将所述步骤(1)的化合物和劳森试剂加入到二氧六环中，高温反应，结束后后经分离提纯，得到噻二唑类化合物；
    (3)将所述步骤(2)的噻二唑类化合物和相应的胺加入到DMSO中反应，结束后经分离提纯，得到中间体化合物；
    (4)将所述步骤(3)的中间体化合物和Pd/C加入到甲醇中，在H2条件下，搅拌反应，结束后经分离提纯，得到含氨基类化合物；
    (5)将所述步骤(4)的含氨基类化合物、相应的醛和NaBH(OAc)3加入到DCM中搅拌反应，结束后经分离提纯，得到含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物。
  4. 根据权利要求3所述的制备方法，其特征在于，所述步骤(1)中4-氯-3-硝基苯甲酸、NH2NHBoc、HBTU和DIEPA的摩尔比为1：1-2：1-2：1-2；所述步骤(2)中化合物和劳森试剂的摩尔比为1：2-3。
  5. 根据权利要求3所述的制备方法，其特征在于，所述步骤(3)中噻二唑类化合物和胺的摩尔比为1：1-2；所述步骤(4)中中间体化合物和Pd/C的质量比为10-12：1；所述步骤(5)中含氨基类化合物、醛和NaBH(OAc)3的摩尔比为1：1-1.5：2-3。
  6. 权利要求1所述的含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物在制备铁死亡抑制剂中的应用。
  7. 权利要求1所述的含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物在制备铁死亡相关疾病的药物中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述铁死亡相关疾病包括神经退行性、组织缺血再灌注损伤、脑卒中、心血管、肝肾衰竭、炎症、糖尿病并发症。
  9. 权利要求1所述的含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物在制备抗氧化荧光探针或抗氧化指示剂中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述抗氧化荧光探针或抗氧化指示剂用于ONOO-高灵敏检测，能够有效的逆转细胞铁死亡进程并且具备自指示能力。
PCT/CN2024/123539 2023-10-23 2024-10-09 一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物及其制备方法和应用 Pending WO2025087033A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202311374155.7A CN117567390A (zh) 2023-10-23 2023-10-23 一种含（1,3,4-噻二唑）苯-1,2-乙二胺类化合物及其制备方法和应用
CN202311374155.7 2023-10-23

Publications (1)

Publication Number Publication Date
WO2025087033A1 true WO2025087033A1 (zh) 2025-05-01

Family

ID=89863316

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/123539 Pending WO2025087033A1 (zh) 2023-10-23 2024-10-09 一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN117567390A (zh)
WO (1) WO2025087033A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117567390A (zh) * 2023-10-23 2024-02-20 中国海洋大学 一种含（1,3,4-噻二唑）苯-1,2-乙二胺类化合物及其制备方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008045358A1 (en) * 2006-10-06 2008-04-17 Trustees Of Princeton Porphyrin catalysts and methods of use thereof
CN116514765A (zh) * 2022-01-24 2023-08-01 华东理工大学 缺血再灌注氧化损伤防治化合物及其制备方法
CN117567390A (zh) * 2023-10-23 2024-02-20 中国海洋大学 一种含（1,3,4-噻二唑）苯-1,2-乙二胺类化合物及其制备方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008045358A1 (en) * 2006-10-06 2008-04-17 Trustees Of Princeton Porphyrin catalysts and methods of use thereof
CN116514765A (zh) * 2022-01-24 2023-08-01 华东理工大学 缺血再灌注氧化损伤防治化合物及其制备方法
CN117567390A (zh) * 2023-10-23 2024-02-20 中国海洋大学 一种含（1,3,4-噻二唑）苯-1,2-乙二胺类化合物及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIU YAWEN: "Development and Bioimaging Application of Fluorescent Probes for Peroxynitrite", THESES, 1 June 2022 (2022-06-01), pages 1 - 87, XP093309456 *

Also Published As

Publication number Publication date
CN117567390A (zh) 2024-02-20

Similar Documents

Publication Publication Date Title
CN111423423A (zh) 一种比率型荧光探针在检测过氧亚硝基阴离子中的应用
Datta et al. A novel chemosensor with visible light excitability for sensing Zn 2+ in physiological medium and in HeLa cells
CN105712964B (zh) 一种基于香豆酰肼的硫醇荧光探针的制备方法及应用
CN110041317A (zh) 一种萘酰亚胺荧光探针及其制备与应用
CN113501818B (zh) 一种荧光探针分子及其制备方法与应用
CN106432257A (zh) 基于罗丹明的gsh水溶性荧光探针的制备方法和应用
CN101948488A (zh) 钌-硒配合物及其在制备荧光探针和抗肿瘤药物中的用途
WO2025087033A1 (zh) 一种含(1,3,4-噻二唑)苯-1,2-乙二胺类化合物及其制备方法和应用
Wang et al. Nanoscale photosensitizer with tumor-selective turn-on fluorescence and activatable photodynamic therapy treatment for COX-2 overexpressed cancer cells
CN111592530B (zh) 含醛肟的他克林衍生物类选择性丁酰胆碱酯酶抑制剂及其制备方法和应用
CN114656476B (zh) 溶酶体靶向性罗丹明b酰肼类荧光探针及制备方法和应用
CN103936724B (zh) 检测组氨酸的荧光探针及其前驱体和它们的制备方法
WO2021104072A1 (zh) 一种过氧化氢响应型Keap1-Nrf2 PPI抑制剂前药、制备方法及其医药用途
CN114805262B (zh) 一种粘度和极性响应型平台荧光探针、硫化氢检测荧光探针及其合成工艺与应用
CN109678848B (zh) 香豆素/吡啶酮杂合衍生物及其制备方法与应用
CN112694471B (zh) 一种苯并吲哚盐-吩噻嗪衍生物及其制备和应用
Panicker et al. A highly lipophilic terpyridine ligand as an efficient fluorescent probe for the selective detection of zinc (II) ions under biological conditions
CN108623611B (zh) 一种检测过氧化氢的荧光探针的合成与应用
CN112341448B (zh) 一种荧光探针及其制备方法与应用
CN115141145A (zh) 一种检测溶酶体次溴酸荧光探针、制备方法与应用
Feng et al. Several Golgi targeting fluorescence markers based on 1, 8-naphthalimide derivatives with amide and long carbon chain
CN117924335A (zh) 一种具有内质网靶向的onoo-荧光探针及其制备方法和应用
CN110804045A (zh) 具潜在抗ad活性的香豆素杂合吡啶酮酰胺衍生物及其制备方法与应用
CN113173940B (zh) 一种双生物活性因子级联激活的抗黑色素瘤前药的合成与应用
CN115572276A (zh) 一种内质网靶向的极性比率型荧光探针及其制备方法和用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24881399

Country of ref document: EP

Kind code of ref document: A1