A (1,3,4-thiadiazole) benzene-1,2-ethanediamine compound, a preparation method and application thereof

Abstract

The application discloses a (1,3,4-thiadiazole) benzene-1,2-ethylenediamine compound and a preparation method and application thereof. The structural formula of the compound is: the compound is verified through experiments, the compound shows strong ferroptosis inhibition activity, good metabolic stability, and is suitable for treatment of ferroptosis related diseases; meanwhile, the structure can be used as a specific ferroptosis probe, has two forms of oxidized state and reduced state, the probe shows green fluorescence in the reduced state, and shows blue fluorescence in the oxidized state, the fluorescence change can be used as an indicator of the degree of action of the antioxidant, and can also be used for detecting the ferroptosis of a to-be-detected sample, especially for ONOO ‑ High-sensitivity detection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, its preparation method, and its application. Background Technology

[0002] Cell death can be broadly classified into two categories: uncontrolled cell death caused by excessive cellular damage and regulated cell death dependent on tightly controlled molecular pathways. Apoptosis is the most typical form of regulated cell death, initiating cell death through the activation of caspases. Ferroptosis, a novel cell death mechanism discovered in recent years, is an oxidative cell death induced by various factors, exhibiting iron ion dependence. Its occurrence stems from an imbalance between the generation and degradation of intracellular lipid reactive oxygen species (ROS). In the early years after its discovery, the mechanisms controlling ferroptosis primarily revolved around cysteine ​​and glutathione metabolism, and the phospholipid peroxidase GPX4's prevention of lipid peroxidation accumulation. Ferroptosis inducers act directly or indirectly on glutathione peroxidases (GPXs) through different pathways, leading to decreased cellular antioxidant capacity, ROS accumulation, and ultimately oxidative cell death. The complex interactions between lipid, iron, and cysteine ​​metabolism have become important regulators of this cell death pathway. Recently, the regulation of ferroptosis has become an attractive strategy for intervening in human diseases, including cancer, neurodegenerative diseases, and ischemic diseases.

[0003] Ferroplasmosis can be inhibited by iron chelators, lipophilic antioxidants, and / or ferrostatin-1 (fer-1). Fer-1, an aryl alkylamine with antioxidant properties, was one of the first feroplasmosis inhibitors identified. As a lipid peroxidation reductant, Fer-1 intercepts and scavenges lipid free radicals through hydrogen atom transfer or direct reduction. However, its short half-life makes further pharmacological evaluation unsuitable.

[0004] In recent years, high-sensitivity, high-resolution fluorescence detection technology has been favored by researchers and widely used in the detection of various substances. Compared with other monitoring methods, fluorescence imaging technology is sensitive, accurate, and can monitor in real time with high accuracy. Therefore, in ferroptosis-related research activities, fluorescent probes are often used to evaluate the process of cellular ferroptosis and the level of action of ferroptosis inhibitors. Nitrosyl peroxide anion (ONOO) - ONOO is an important oxide species, and studies have shown that it is closely related to ferroptosis. Many chromophore-based fluorescent probes have been applied to ONOO in vivo. -Recognition, such as of rhodamine and nilored compounds. However, these small molecules only have recognition functions and are not suitable for use as antioxidant drugs, thus still having certain limitations in practical applications.

[0005] Typically, fluorescent probes detect substances by increasing or decreasing fluorescence intensity. Based on this, a probe capable of detecting substances with ONOO is designed. - A fluorescent probe for the reaction, which can consume ONOO through a chemical reaction. - This allows them to exert antioxidant effects, and the changes in fluorescence spectra before and after the reaction enable real-time monitoring. Such self-indicating fluorescent antioxidant probes can self-monitor the process of reversing ferroptosis without requiring additional sample processing, providing a novel approach for evaluating ferroptosis antioxidants. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, its preparation method, and its applications. The compound exhibits strong antioxidant activity and good metabolic stability, making it suitable for the treatment of ferroptosis-related diseases; it also displays significant fluorescence spectral changes, particularly concerning ONOO. - It exhibits extremely high selectivity, with obvious and easily identifiable phenomena.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] On one hand, the present invention provides a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, the structural formula of which is shown below:

[0009]

[0010] In the formula, R 1 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-C 10 Aromatic heterocyclic groups or C3-C7 saturated heterocyclic groups;

[0011] R 2 Selected from C0-C8 alkyl, C3-C 12 Cycloalkyl, adamantyl, or polyacetylenic;

[0012] R 3 Selected from hydrogen, alkyl, aryl, C1-C6 alkyl-aryl, C1-C6 alkyl-phenolic or C3-C4 10 cycloalkyl;

[0013] Among them, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkoxy, phenyl, benzyl, naphthyl, C5-C 10 Aromatic heterocyclic groups, C3-C7 saturated heterocyclic groups, C3-C 12 Cycloalkyl, polyynyl, aryl, C1-C6 alkyl-aryl, C1-C6 alkyl-phenolic or C3-C 10 Cycloalkyl groups can be substituted by one or more atoms or groups;

[0014] n is the number of flexible alkyl chains, n = 0 or 2-4.

[0015] Preferably, the R 1 It is selected from C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C3-C8 cycloalkoxy, aryl and derivatives.

[0016] Preferably, the R 2 Selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and their derivatives, cycloheptyl, adamantyl and their derivatives.

[0017] Preferably, the R 3 Selected from cyclopropyl, cyclopentyl, cyclohexyl, arylbenzyl and their derivatives.

[0018] Preferably, n = 0, 2-4.

[0019] Preferably, the compounds are specifically compounds I-1, II-1 to II-9, III-1 to III-15, and IIII-1 to IIII-12, with the following specific structural formulas:

[0020]

[0021]

[0022] On the other hand, the present invention also provides a method for preparing the aforementioned (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compounds, which includes the following steps:

[0023]

[0024] (1) Compound 4-chloro-3-nitrobenzoic acid, NH2NHBoc, HBTU and DIEPA were added to DMF and reacted at room temperature for 4 hours. After the reaction was completed, the mixture was separated and purified to obtain a pale yellow solid compound 2.

[0025] (2) Compound 2 from step (1) and Lawson's reagent were added to dioxane and reacted overnight at 110°C. After the reaction was completed, the thiadiazole compound 3 was obtained by separation and purification.

[0026] (3) The thiadiazole compound 3 and the corresponding amine from step (2) were added to DMSO and reacted at 60°C for 18 hours. After the reaction was completed, the intermediate compound 4 was obtained by separation and purification.

[0027] (4) The intermediate compound 4 and Pd / C from step (3) were added to methanol and stirred at room temperature for 6 hours under H2 conditions. After the reaction was completed, the mixture was separated and purified to obtain amino-containing compound 5.

[0028] (5) The amino-containing compound 5, the corresponding aldehyde and NaBH(OAc)3 from step (4) are added to DCM and stirred at room temperature for 6 hours. After the reaction is completed, the mixture is separated and purified to obtain (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound 6.

[0029] Preferably, in step (1), the molar ratio of 4-chloro-3-nitrobenzoic acid, NH2NHBoc, HBTU and DIEPA is 1:1.5:1.5:1.5.

[0030] Preferably, the molar ratio of compound 2 and Lawson's reagent in step (2) is 1:2.5.

[0031] Preferably, in step (3), the molar ratio of thiadiazole compound 3 to the corresponding amine is 1:1.5.

[0032] Preferably, in step (4), the mass ratio of intermediate compound 4 to Pd / C is 10:1.

[0033] Preferably, in step (5), the molar ratio of amino compound 5, the corresponding aldehyde, and NaBH(OAc)3 is 1:1.1:2.

[0034] Preferably, the specific method for separation and purification in step (1) is as follows: after the reaction is completed, the mixture 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 pale yellow solid compound 2.

[0035] Preferably, the specific method for separation and purification in step (2) is as follows: after the reaction is completed, the mixture is cooled to room temperature, the solvent is evaporated, the mixture is extracted with water and ethyl acetate, the organic layers are combined, and the combined organic layers are washed with 10% sodium bicarbonate solution. Then the organic layers are dried with sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain thiadiazole compound 3.

[0036] Preferably, the specific method for separation and purification in step (3) is as follows: after the reaction is completed, the mixed reaction 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 intermediate compound 4.

[0037] Preferably, the specific method for separation and purification in step (4) is as follows: 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 amino-containing compound 4;

[0038] Preferably, the specific method for separation and purification in step (5) is as follows: 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, then washed with brine, dried, filtered, vacuum concentrated, and the mixture is separated by column chromatography to obtain compounds containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine.

[0039] On the other hand, the present invention also provides the application of the aforementioned (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compounds in the preparation of ferroptosis inhibitors.

[0040] On the other hand, the present invention also provides the use of the aforementioned (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compounds in the preparation of medicaments for ferroptosis-related diseases.

[0041] Furthermore, the iron death-related diseases include neurodegenerative diseases, tissue ischemia-reperfusion injury, stroke, cardiovascular diseases, liver and kidney failure, inflammation, and diabetic complications.

[0042] Furthermore, the drug contains a (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound or a pharmaceutically acceptable salt thereof, excipient, or carrier.

[0043] On the other hand, the present invention also provides the application of the aforementioned (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compounds in the preparation of antioxidant fluorescent probes or antioxidant indicators.

[0044] Furthermore, the antioxidant fluorescent probe or antioxidant indicator is used in ONOO - It has high sensitivity, can effectively reverse the process of ferroptosis, and has self-indication capabilities.

[0045] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0046] 1. This invention designs and obtains 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 research on ferroptosis-related diseases;

[0047] 2. The preparation method of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound described in this invention is simple, has a high yield, and is suitable for large-scale application.

[0048] 3. The (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound described in this invention can be used as the fluorescent probe, exhibiting significant changes in fluorescence spectrum. During the inhibition of ferroptosis, the effect of the probe can be evaluated and monitored in real time by the changes in its own fluorescence spectrum. Attached Figure Description

[0049] Figure 1A The 1H NMR spectrum of compound I-1; Figure 1B The carbon spectrum of compound I-1; Figure 1C High-resolution mass spectra of compound I-1;

[0050] Figure 2 Detection of ONOO by Antioxidant Fluorescent Probe I-1 - The selective test results are shown in the figure, where 1-14 represent the bioactive small molecules Blank and Cu, respectively. 2+ Zn 2+ Fe 3+ Fe 2+ Al 3+ Ca 2+ SO4 2- SO3 2- NO2 - O 2- ,t-BuOOH,H2O2,HClO,ONOO - ,·OH,Vc - GSH, VC;

[0051] Figure 3 Detection of ONOO by Antioxidant Fluorescent Probe I-1 - The fluorescence spectrum results are shown in the figure.

[0052] Figure 4 The fluorescence spectra of the antioxidant fluorescent probe I-1 in different solvents are shown in the figure.

[0053] Figure 5 Figure showing the results of flow cytometry assessment of I-1 antioxidant activity and its self-indicating ability. Detailed Implementation

[0054] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0055] The overall synthetic route for a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine is as follows:

[0056]

[0057] Among them, (a)HBTU,DIEPA,NH2NHCOR 1 (b) Lloyd's reagent, reflux; (c) the corresponding amine, K2CO3, DMSO, 80℃; (d) Pd / C, H2, CH3OH; (e) the corresponding aldehyde, NaBH(OAc)3, DCM.

[0058] (1) Synthesis method a: Compound 1 (4.96 mmol), NH2NHCOR 1 7.44 mmol of HBTU and DIEPA were added to a reaction flask containing 3 mL of DMF and reacted at room temperature until complete. The mixture was poured into 30 mL of water, the aqueous layer was extracted with ethyl acetate, the organic layer was dried and concentrated, and the mixture was separated by column chromatography to obtain a pale yellow solid 2.

[0059] (2) Synthesis method b: Compound 2 (0.78 mmol) obtained in the previous step and Lawson's reagent (1.94 mmol) were added to a reaction flask, with dioxane as the solvent, and the reaction was carried out overnight at 110 °C. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated, and the mixture was extracted with water and ethyl acetate. The organic layers were combined and washed with 10% sodium bicarbonate solution. The organic layers were then dried with sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain a yellow solid, which was thiadiazole compound 3.

[0060] (3) Synthesis method c: Compound 3 (0.45 mmol) obtained in the previous step and the corresponding amine (0.67 mmol) were added to a reaction flask, with DMSO (3 mL) as the solvent. The reaction solution was reacted at 60 °C until the reaction was detected by TLC. The mixed reaction solution was poured into 30 mL of water, the aqueous layer was extracted with ethyl acetate, the organic layer was dried and concentrated, and the mixture was separated by column chromatography to obtain a yellow solid 4.

[0061] (4) Synthesis method d: Compound 4 (0.35 mmol) was dissolved in methanol and reacted with 10% Pd / C under H2 conditions at room temperature for 6 hours. The solution was filtered through a diatomaceous earth pad and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to obtain the desired compound 5.

[0062] (5) Synthetic method e: Compound 5 (0.2 mmol) was added to a DCM solution containing benzaldehyde (0.23 mmol) and stirred at room temperature for 30 minutes. Then, sodium triacetoxyborohydride (0.4 mmol) was added in a single batch, and the reaction mixture was stirred for 24 hours. The aqueous layer was then extracted with CH2Cl2 from a saturated sodium bicarbonate solution. The combined organic layers were washed with saturated sodium bicarbonate solution, then washed with brine, dried, filtered, concentrated under vacuum, and the mixture was separated by column chromatography to obtain the target compound 6.

[0063] Example 1: Preparation of compound I-1

[0064]

[0065] In this embodiment, compound I-1 was prepared using the experimental method described above. 1 H 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). 13 C 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 C 15 H 20 N4S[M+H] + calcd 289.1487, found 289.1481 (Figure 1).

[0066] Example 2: Preparation of compounds II-1 to II-9

[0067] In this embodiment, compounds II-1 to II-9 were prepared using the experimental method described above:

[0068]

[0069] 1 H 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). 13 C NMR (101MHz, DMSO-d6) δ169.82,162.79,140.46,135.41,118.98,118.18,112.14,108.87,30.30,15.65.

[0070]

[0071] 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). 13 C 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.

[0072]

[0073] 1 H 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). 13 C 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.

[0074]

[0075] 1 H 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). 13 C 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.

[0076]

[0077] 1 H 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). 13 C 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 C 14 H 18 N4S[M+H] + calcd 275.1330,found275.1325.

[0078]

[0079] 1 H 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). 13 C 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 C 16 H 22 N4S[M+H] + calcd303.1643,found303.1638.

[0080]

[0081] 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). 13 C 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.

[0082]

[0083] 1 H 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). 13 C 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.

[0084]

[0085] 1 H 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)δ169.42,163.24,154.77,139.09,133.77,121.90,1 20.18,116.13,111.48,79.74,50.02,32.13,29.70,28.44,15.72.HRMS(ESI)for C 19 H 27 N5O2S[M+H] + calcd 390.1964, found 390.1958.

[0086] Example 3: Preparation of compounds III-1 to III-15

[0087] In this embodiment, compounds III-1 to III-15 were prepared using the experimental methods described above:

[0088]

[0089] 1 H 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). 13 C NMR(101MHz,Chloroform-d)δ170.00,162.90,139.80,139.00,136.22,128.68,128.15,1 27.48,121.13,111.61,110.60,51.60,49.20,33.38,29.70,25.89,24.99.HRMS(ESI)for C 22 H 26 N4S[M+H] + calcd 379.1956,found379.1951.

[0090]

[0091] 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). 13 C 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 C 22 H 25 N5S[M+H] + calcd 380.1909,found 380.1903.

[0092]

[0093] 1 H 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). 13 C 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.

[0094]

[0095] 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). 13 C 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.

[0096]

[0097] 1 H 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). 13 C 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 C 22 H 25 FN4S[M+H] + calcd 397.1862,found 397.1857.

[0098]

[0099] 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). 13 C 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.

[0100]

[0101] 1 H 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). 13 C 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.

[0102]

[0103] 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). 13 C 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 C 20 H 24 N4S2[M+H] + calcd 385.1521,found385.1515.

[0104]

[0105] 1 H 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(m 1H),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). 13 C 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.

[0106]

[0107] 1 H 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). 13 C 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.

[0108]

[0109] 1 H 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). 13 C 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.

[0110]

[0111] 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). 13 C 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.

[0112]

[0113] 1 H 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). 13 C 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.

[0114]

[0115] 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,2 H),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). 13 C 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.

[0116]

[0117] 1 H 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). 13 C 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.

[0118] Example 4: Preparation of compounds IIII-1 to IIII-11

[0119] In this embodiment, compounds IIII-1 to IIII-11 were prepared using the experimental method described above:

[0120]

[0121] 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). 13 C 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.

[0122]

[0123] 1 H 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). 13 C 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.

[0124]

[0125] 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). 13 C 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.

[0126]

[0127] 1 H 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). 13 C 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.

[0128]

[0129] 1 H 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.

[0130]

[0131] 11 H 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). 13 C 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.

[0132]

[0133] 1 H 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). 13 C 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.

[0134]

[0135] 1 H 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). 13 C 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.

[0136]

[0137] 1 H 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). 13 C 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.

[0138]

[0139] 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). 13 C 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.

[0140]

[0141] 1 H 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),3.3 6-3.26(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). 13 C NMR(101MHz,Chloroform-d)δ172.25,153.95,153.56,153.18,152.79,140.63,134.8 7,120.74,116.48,111.88,109.20,54.08,50.54,32.58,32.29,24.86,23.95,23.35.

[0142] Example 5: Preparation of compound IIII-12

[0143] In this embodiment, compound IIII-12 was prepared using the experimental method described below:

[0144]

[0145] Among them, (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.

[0146] (1) Synthesis of methyl 1,2,3,4-tetrahydroquinoline-6-carboxylate (compound 2)

[0147]

[0148] Methyl quinoline-6-carboxylate (0.3 g, 1.6 mmol) was added to a reaction flask, and glacial acetic acid (20 mL) was used as a solvent. NaBH3CN (0.302 g, 0.48 mmol) was added in portions under ice bath conditions. The reaction was then carried out at room temperature until completion. The reaction solution was poured into a saturated aqueous solution of NaHCO3, extracted with ethyl acetate, and the organic layers were combined and dried. The mixture was concentrated under vacuum and then separated by column chromatography to obtain a white solid (0.16 g, 52.28%).

[0149] 1 H 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).

[0150] (2) Synthesis of 3-dihydroquinoline-1,6(2H)-dicarboxylic acid 1-benzyl-6-methyl (compound 3)

[0151]

[0152] Methyl 1,2,3,4-tetrahydroquinoline-6-carboxylate (0.25 g, 1.3 mmol) and DIEPA (0.67 g, 5.2 mmol) were dissolved in a reaction flask containing 20 mL of dichloromethane. Cb2-Cl (0.67 g, 3.9 mmol) was added dropwise under ice bath conditions, and the reaction was carried out at room temperature until completion. The mixture was separated by column chromatography to obtain the target compound (0.39 g, 91.7%) as a white solid.

[0153] 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).

[0154] (3) Synthesis of 8-nitro-3,4-dihydroquinoline-1,6(2H)-dicarboxylic acid 1-benzyl-6-methyl (compound 4)

[0155]

[0156] 0.3 g (0.92 mmol) of 3-dihydroquinoline-1,6(2H)-dicarboxylic acid-1-benzyl-6-methyl was dissolved in a reaction flask containing 5 mL of trifluoroacetic acid. NaNO2 (0.07 g, 1.01 mmol) was added in portions under ice bath conditions. The reaction was carried out for 1 h under ice bath conditions, followed by 2 h at room temperature. After the reaction was complete, the mixture was poured into an aqueous ammonia solution, and the aqueous layer was extracted with EA. The organic layers were combined and dried. The mixture was concentrated under vacuum, and then separated by column chromatography to obtain a yellow oil (0.21 g, 61.76%).

[0157] 1 H NMR(400MHz,DMSO-d6)δ8.21(d,J=1.9Hz,1H),8.07(d,J=2.0Hz,1H),7.47-7.1 9(m,5H),5.31-5.02(m,2H),3.88(s,5H),3.05-2.72(m,2H),2.11-1.65(m,2H).

[0158] (4) Synthesis of 6-(5-methyl-1,3,4-thiadiazol-2-yl)-8-nitro-3,4-dihydroquinoline-1(2H)-carboxylic acid benzyl ester (compound 7)

[0159]

[0160] 6-(2-acetylhydrazine-1-carbonyl)-8-nitro-3,4-dihydroquinoline-1(2H)-carboxylic acid benzyl ester (0.5 g, 1.21 mmol) and Lawson's reagent (2.93 g, 7.26 mmol) were added to a reaction flask, with dioxane as the solvent (50 mL), and the mixture was refluxed overnight. The solvent was evaporated under vacuum, and the remaining mixture was dissolved in ethyl acetate. The organic layer was washed with a saturated sodium bicarbonate solution, and the organic layers were combined and concentrated. The mixture was separated by column chromatography to give a yellow oil (0.25 g, 50.3%).

[0161] 1 H 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).

[0162] (5) Synthesis of compound IIII-12

[0163]

[0164] Compound 7 (0.2 g, 0.48 mmol) was added to a reaction flask containing 20 mL of methanol solution, and 10% palladium on carbon (0.04 g) was added. The reaction was carried out under hydrogen atmosphere for 12 h. After the reaction was completed, the palladium on carbon was filtered off with diatomaceous earth, and the mixture was concentrated and then subjected to column chromatography to obtain compound IIII-12 (0.07 g, 58.33%).

[0165] 1 H 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). 13 C 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.

[0166] Example 6: Test of the compound's ferroptosis inhibitory activity

[0167] Studies have shown that GPX4 inhibitors, such as RSL-3, can induce ferroptosis in cells, which can be blocked by other small molecules, such as lipophilic antioxidants like Ferrostatin-1 (fer-1) and Liproxstatin. Therefore, the ability of ferroptosis inhibitors to block ferroptosis can be indicated by the reversal of ferroptosis induced by ferroptosis inducers.

[0168] Cell lines: human renal cell carcinoma cell line OS-RC-2 and human neuroblastoma cell line SH-SY5Y, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0169] Methods: The MTT assay was used, specifically as follows: Human renal cell carcinoma line OS-RC-2 / human neuroblastoma cells SH-SY5Y in logarithmic growth phase were digested, collected, and diluted. Approximately 4000-5000 cells per well were seeded into 96-well plates, with three replicates and 80 μL per well. The plates were incubated overnight at 37°C with 5% CO2. The experiment included a DMSO control group and nine different concentrations of the compound treatment groups. Different concentrations of the compound were added to each treatment group, and a DMSO control group (diluted to the highest concentration compound) was set up. After incubation for 1 hour at 37°C with 5% CO2, MRSL3 at a concentration of 5 μL was added to each compound concentration to induce ferroptosis. RSL3 and DMSO control groups were also set up. The plates were incubated for another 48 hours at 37°C with 5% CO2. Add 20 μL of 5 mg / mL MTT solution to each well and incubate OS-RC-2 cells at 37°C for 2 h. Then add 100 μL of DMSO to each well, shake gently for 10 min, and mix well. Measure the optical density (OD value) of each well at 570 nm using an ELISA reader. Repeat the experiment three times. Calculate the survival rate (%) using the following formula: Survival rate % = (OD value of experimental group / OD value of DMSO control group) × 100%.

[0170] The results are shown in Table 1, indicating that the various compounds of the present invention can significantly inhibit ferroptosis and have good ferroptosis inhibitory activity.

[0171] Table 1: Results of the ferroptosis inhibitory activity test of the compounds

[0172]

[0173]

[0174]

[0175]

[0176] In the table: "A" indicates IC 50 ≤100nM, “B” indicates IC 50 >100nM and ≤500nM, "C" indicates IC 50 >500nM and ≤1μM, “D” indicates IC 50 >1μM and ≤10μM.

[0177] Example 7: In vitro / intracellular drug metabolism properties of the compound

[0178] In vitro assay: Plasma and liver microsome homogenate samples were stored at -20°C and gradually warmed to room temperature before use. The compound was dissolved in acetonitrile to prepare a 4 mg / mL stock solution, which was then added to the plasma or liver microsome homogenate to a final concentration of 0.4 mg / mL. The solutions were incubated at 37°C for the required times (0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 24 h). The incubation was terminated with acetonitrile at the end of the incubation period. The mixture was vortexed for 30 s and centrifuged at 12,000 rpm for 10 min, then filtered through a 0.22 μM filter. The filtrate was analyzed using an Agilent 1260 HPLC system with an Alltima C18 column (5 μm, 4.6 mm × 250 mm). The flow rate was maintained at 400 μL / min, with initial flow conditions of 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% within 0.50 min, held for 1.50 min, then increased to 100% within 5.00 min and held for 3 min. Next, within 0.50 min, solvent B was reduced to the initial condition (20%), with a total run time of 12.00 min. The percentage of compound remaining at each time point was equal to the ratio of the compound peak integral to the internal standard peak integral, with the internal standard peak area being the peak area at time t=0.

[0179] The results are shown in Table 2 below. The thiadiazole compounds based on the Fer-1 structure of this invention exhibit good stability in in vitro plasma and liver microsomes, and have excellent ADME properties, which can be used for in vivo bioactivity studies.

[0180] Table 2: ADME property test results

[0181]

[0182] In vivo assay: After a single intravenous injection of the compound into male mice, blood and brain tissue samples were collected at 0, 0.5, 2, 4, and 8 hours. The concentrations of the compound in mouse plasma and brain tissue were determined by LC-MS / MS, and relevant pharmacokinetic parameters were calculated to investigate the pharmacokinetic characteristics and brain tissue distribution of the compound in mice. The results showed that compound I-1 exhibited good metabolic stability in mice, with a plasma half-life of 2.28 hours. Furthermore, it can cross the blood-brain barrier, further indicating that this type of compound can be used in in vivo studies for the treatment of diseases related to ferroptosis.

[0183] Table 3: Pharmacokinetic parameters of I-1

[0184]

[0185] Example 8: The compound can be used as a fluorescent probe to detect ferroptosis.

[0186] Detection of ONOO by Antioxidant Fluorescent Probe I-1 - Selectivity testing: Under the same test conditions, excess amounts of other bioactive small molecules were added to the solution, and fluorescence spectra were measured. The excitation wavelength was 350 nm, and the maximum emission wavelengths were 436 nm and 525 nm, respectively. Results are as follows: Figure 2 As shown, 1-14 represent the bioactive small molecules Blank and Cu, respectively. 2+ Zn 2+ Fe 3+ Fe 2 + Al 3+ Ca 2+ SO4 2- SO3 2- NO2 - O 2- ,t-BuOOH,H2O2,HClO,ONOO - ,·OH,Vc - GSH, VC. F 436 / F 525 Only ONOO - The fluorescence is significantly enhanced in the presence of ONOO, and other bioactive small molecules do not interfere with the detection results, indicating that the fluorescent probe prepared in this invention is effective against ONOO. - It has a high degree of selectivity.

[0187] Detection of ONOO by Antioxidant Fluorescent Probe I-1 - Fluorescence spectrum: A fluorescent probe with an initial concentration of 1 mM was added to PBS buffer (pH = 7.4, 0.5% DMSO) to bring the concentration of the fluorescent probe in the solution to 10 μM. Then, different amounts of ONOO with an initial concentration of 10 mM were added sequentially. - Not joining ONOO - As a control, ONOO was 37℃ for 90 minutes. - The sample reacted fully with the fluorescent probe. Different concentrations of ONOO were tested using a fluorescence spectrometer. - The fluorescence spectrum under the specified conditions, with an excitation wavelength of 350 nm and emission wavelengths of 436 nm and 525 nm. For example... Figure 3 As shown, with ONOO - With increasing concentration, the fluorescence intensity at 436 nm gradually increases, while the fluorescence intensity at 525 nm gradually decreases.

[0188] Fluorescence spectra of antioxidant fluorescent probe I-1 in different solvents: An initial concentration of 1 mM of the fluorescent probe was added to different solvents to achieve a concentration of 20 μM. The fluorescence spectra in these solvents were then measured using a fluorescence spectrometer. The excitation wavelength was 350 nm, and the emission wavelength was 525 nm. Figure 4 As shown, the maximum emission wavelength of I-1 gradually increases with increasing solvent polarity.

[0189] Flow cytometry was used to assess the antioxidant activity of I-1 and its self-indicating ability: OS-RC2 cells were incubated with probe I-1, while the control group was incubated with the antioxidant Fer-1. Cells were cultured in a humid environment at 37°C with 5% CO2. Then, the cells were incubated with RSL3 for 1 h, with the RSL3 group serving as a reference control. Finally, BODIPY was used... 581 / 591 After incubating the cells for 30 minutes, flow cytometry was performed. Untreated cells served as a blank control. Figure 5 As can be seen from this, compared with the RSL3 group, the I-1 treated cells were able to effectively reverse the process of ferroptosis, which was basically consistent with the results of the Fer-1 control group. In addition, the I-1 channel showed obvious fluorescence changes. Figure 5 b) This demonstrates that the antioxidant fluorescent probe prepared in this invention can effectively reverse the process of ferroptosis and has self-indicating ability.

[0190] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, characterized in that, The compounds are specifically compounds I-1, II-1~II-9, III-1~III-15, and IIII-1~IIII-12, and their structural formulas are as follows: ， ， ， 。 2. The method for preparing the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound according to claim 1, characterized in that, The preparation method includes the following steps: ， (a) 4-chloro-3-nitrobenzoic acid, NH2NHCOR 1 HBTU and DIEPA were added to DMF and reacted. After the reaction was completed, the mixture was separated and purified to obtain a pale yellow solid compound 2. (b) Compound 2 from step (a) and Lawson's reagent were added to dioxane and reacted at high temperature. After the reaction was completed, the mixture was separated and purified to obtain thiadiazole compound 3. (c) The thiadiazole compound 3 and the corresponding amine from step (b) are added to DMSO for reaction. After the reaction is completed, the intermediate compound 4 is obtained by separation and purification. (d) The intermediate compound 4 from step (c) and Pd / C were added to methanol and stirred under H2 conditions. After the reaction was completed, the mixture was separated and purified to obtain amino-containing compound 5. (e) The amino-containing compound 5 from step (d), the corresponding aldehyde and NaBH(OAc)3 were added to DCM and stirred to react. After the reaction was completed, the mixture was separated and purified to obtain compound 6 containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine. In the reaction formula, compound 6 refers to specific compounds I-1, II-1~II-9, III-1~III-15, and IIII-1~IIII-11, and the substituent R in compound 6 is... 1 R 2 R 3 These are the corresponding substituents in the specific compounds.

3. The preparation method according to claim 2, characterized in that, In step (a), 4-chloro-3-nitrobenzoic acid and NH2NHCOR 1 The molar ratio of HBTU and DIEPA is 1:1-2:1-2:1-2; the molar ratio of the compound and Lawson's reagent in step (b) is 1:2-3.

4. The preparation method according to claim 2, characterized in that, In step (c), the molar ratio of thiadiazole compound to amine is 1:1-2; in step (d), the mass ratio of intermediate compound to Pd / C is 10-12:1; in step (e), the molar ratio of amino compound, aldehyde and NaBH(OAc)3 is 1:1-1.5:2-3.

5. The use of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound of claim 1 in the preparation of ferroptosis inhibitors.

6. The use of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound as described in claim 1 in the preparation of a medicament for ferroptosis-related diseases.

7. The application according to claim 6, characterized in that, The iron death-related diseases mentioned include neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, liver and kidney failure, inflammation, and diabetic complications.

8. The (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound of claim 1 in the preparation and detection of ONOO - Applications in antioxidant fluorescent probes or antioxidant indicators.

9. The application according to claim 8, characterized in that, The antioxidant fluorescent probe or antioxidant indicator is used in ONOO - It has high sensitivity, can effectively reverse the process of ferroptosis, and has self-indication capabilities.

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