Compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, a preparation method therefor and a use thereof. The compound has a structural formula. According to the present invention, upon experimental verification, the compound demonstrates potent ferroptosis inhibitory activity and good metabolic stability, and is suitable for the treatment of ferroptosis-related diseases; and the structure can be used as a probe for specific ferroptosis and has two forms of an oxidation state and a reduction state, the probe exhibits green fluorescence in the reduction state, and exhibits blue fluorescence in the oxidation state, and the fluorescence change of the probe can be used as an indicator of the degree of action of an antioxidant, and can be used for detecting the progress of ferroptosis in a sample to be tested, especially for high-sensitivity detection of ONOO-.

Description

A compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, and its preparation method and application Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, a preparation method and an application thereof. Background Art

[0002] Cell death can be broadly divided into two categories: uncontrolled cell death caused by excessive cellular damage and regulated cell death that relies 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 newly discovered form of cell death, is an oxidative cell death induced by various factors and is iron-dependent. Its occurrence is caused by an imbalance in the generation and degradation of reactive oxygen species (ROS) in intracellular lipids. The mechanisms controlling ferroptosis elucidated in the initial years of its discovery primarily revolved around cysteine ​​and glutathione metabolism, as well as the prevention of lipid peroxidation accumulation by the phospholipid peroxidase GPX4. Ferroptosis inducers act directly or indirectly on glutathione peroxidases (GPXs) through diverse pathways, leading to reduced cellular antioxidant capacity, ROS accumulation, and ultimately oxidative cell death. The complex interplay between lipid, iron, and cysteine ​​metabolism has emerged as a key regulator of this cell death pathway. Recently, modulation of ferroptosis has emerged as an attractive strategy for intervention in human diseases, including cancer, neurodegenerative disorders, and ischemic diseases.

[0003] Ferroptosis can be inhibited by iron chelators, lipophilic antioxidants, and / or ferrostatin-1 (fer-1). Fer-1, an arylalkylamine with antioxidant properties, 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.

[0004] In recent years, high-sensitivity, high-resolution fluorescence detection technology has been favored by researchers and has been widely used in the detection of various substances. Compared with other monitoring methods, fluorescence imaging technology is sensitive and accurate, can be monitored in real time with high accuracy. Therefore, in ferroptosis-related scientific research activities, fluorescent probes are often used to evaluate the process of cell ferroptosis and the level of action of ferroptosis inhibitors. Peroxynitrosyl anion (ONOO -) is an important oxidizing species. Studies have shown that it is closely related to ferroptosis. Many chromophore-based fluorescent probes have been used to detect ONOO in organisms. - However, these small molecules only have recognition functions and are not suitable for use as antioxidant drugs, so they still have certain limitations in practical applications.

[0005] Generally, fluorescent probes detect substances by increasing or decreasing the fluorescence intensity. Based on this, a method that can be used with ONOO - A fluorescent probe that can consume ONOO through chemical reactions - This allows for real-time monitoring of the ferroptosis-reversing process, while also providing a novel approach for developing ferroptosis-reversing antioxidants based on fluorescence spectra changes before and after the reaction.

[0006] Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention provides a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine and its preparation method and application. 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, and has a strong effect on ONOO - It has strong selectivity, obvious phenomenon and is easy to identify.

[0008] The object of the present invention is achieved through the following technical solutions:

[0009] In one aspect, the present invention provides a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, the structural formula of which is shown below:

[0010] Where 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 group or C3-C7 saturated heterocyclic group;

[0011] R 2 Selected from C0~C8 alkyl, C3~C 12 cycloalkyl, adamantyl or polyalkynyl;

[0012] R 3 Selected from hydrogen, alkyl, aryl, C1-C6 alkyl-aryl, C1-C6 alkyl-phenol or C3-C10 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 group, C3-C7 saturated heterocyclic group, C3~C 12 Cycloalkyl, polyalkynyl, aryl, C1-C6 alkyl-aryl, C1-C6 alkyl-phenol or C3-C 10 Cycloalkyl groups can be substituted with 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 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 its derivatives, cycloheptyl, adamantyl and its derivatives.

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

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

[0019] Preferably, 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:

[0020] On the other hand, the present invention also provides a method for preparing the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound, which comprises the following steps:

[0021] (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, the mixture was separated and purified to obtain a light yellow solid compound 2;

[0022] (2) adding the compound 2 prepared in step (1) and Lawesson's reagent to dioxane, reacting at 110° C. overnight, and separating and purifying the mixture after the reaction to obtain a thiadiazole compound 3;

[0023] (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 the reaction to obtain the corresponding intermediate compound 4;

[0024] (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 the amino-containing compound 5;

[0025] (5) The amino compound 5 of step (4), the corresponding aldehyde and NaBH(OAc)3 were added to DCM, stirred at room temperature for 6 hours, and the reaction was completed and then separated and purified to obtain the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound 6.

[0026] 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.

[0027] Preferably, the molar ratio of compound 2 to Lawesson's reagent in step (2) is 1:2.5.

[0028] Preferably, in step (3), the molar ratio of the thiadiazole compound 3 to the corresponding amine is 1:1.5.

[0029] Preferably, in step (4), the mass ratio of the intermediate compound 4 to Pd / C is 10:1.

[0030] Preferably, the molar ratio of the amino compound 5, the corresponding aldehyde and NaBH(OAc)3 in step (5) is 1:1.1:2.

[0031] Preferably, 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.

[0032] 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. The organic layer is then dried over sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain thiadiazole compound 3.

[0033] Preferably, 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.

[0034] Preferably, 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;

[0035] Preferably, the specific method for 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.

[0036] On the other hand, the present invention also provides the use of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine-containing compound in the preparation of ferroptosis inhibitors.

[0037] On the other hand, the present invention also provides the use of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound in the preparation of drugs for ferroptosis-related diseases.

[0038] Furthermore, the ferroptosis-related diseases include neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, liver and kidney failure, inflammation, and diabetic complications.

[0039] Furthermore, the drug contains a (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound or a pharmaceutically acceptable salt thereof, an excipient or a carrier.

[0040] On the other hand, 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.

[0041] Furthermore, the antioxidant fluorescent probe or antioxidant indicator is used for ONOO - Highly sensitive detection, can effectively reverse the process of cell ferroptosis and has self-indication capabilities.

[0042] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0043] 1. 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;

[0044] 2. The preparation method of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound of the present invention is simple, has high yield, and is suitable for large-scale promotion and application;

[0045] 3. 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. During the process of inhibiting ferroptosis, the effect of the monitoring effect can be evaluated in real time through changes in its own fluorescence spectrum. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1A is a hydrogen nuclear magnetic resonance spectrum of compound I-1; Figure 1B is a carbon spectrum of compound I-1; Figure 1C is a high-resolution mass spectrum of compound I-1;

[0047] Figure 2 shows the detection of ONOO by antioxidant fluorescent probe I-1 - Selectivity test results, where 1-14 represent the biologically active small molecules Blank, Cu 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;

[0048] Figure 3 shows the detection of ONOO by antioxidant fluorescent probe I-1 - Fluorescence spectrum results of ;

[0049] FIG4 is a graph showing the fluorescence spectra of the antioxidant fluorescent probe I-1 in different solvents;

[0050] FIG5 is a graph showing the results of flow cytometry evaluation of the antioxidant activity of I-1 and its self-indicating ability. DETAILED DESCRIPTION

[0051] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, the protection scope of the present invention is not limited to the following embodiment.

[0052] The overall synthetic route of a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine is as follows:

[0053] Among them, (a) HBTU, DIEPA, NH2NHCOR 1; (b) Lawesley's reagent, reflux; (c) corresponding amine, K2CO3, DMSO, 80℃; (d) Pd / C, H2, CH3OH; (e) corresponding aldehyde, NaBH(OAc)3, DCM.

[0054] (1) Synthesis method a: Compound 1 (4.96 mmol), NH2NHCOR 1 (7.44 mmol), HBTU (7.44 mmol), and DIEPA (7.44 mmol) were added to a reaction flask containing DMF (3 mL) and reacted at room temperature until completion. The mixture was poured into 30 mL of water, and 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 light yellow solid 2.

[0055] (2) Synthesis method b: Compound 2 (0.78 mmol) obtained in the previous step and Lawesson's reagent (1.94 mmol) were added to a reaction flask, dioxane was used as the solvent, and the reaction was carried out at 110°C overnight. 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 then washed with 10% sodium bicarbonate solution. The organic layer was then dried over sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain a yellow solid, which was thiadiazole compound 3.

[0056] (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 solvent. The reaction solution was reacted at 60°C until the reaction was complete as determined by TLC. The mixed reaction solution was poured into 30 mL of water, and 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.

[0057] (4) Synthesis Method d: Compound 4 (0.35 mmol) was dissolved in methanol and reacted with 10% Pd / C under H2 at room temperature for 6 hours. The solution was filtered through a pad of Celite, and the solvent was removed under reduced pressure. The residue was purified by silica gel flash column chromatography to yield the desired compound 5.

[0058] (5) Synthesis 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. Sodium triacetoxyborohydride (0.4 mmol) was then added in one portion and the reaction was stirred for 24 hours. The mixture was then poured into a saturated aqueous sodium bicarbonate solution and the aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with a saturated aqueous sodium bicarbonate solution and then with brine, dried, filtered, and concentrated in vacuo. The mixture was separated by column chromatography to obtain the target compound 6.

[0059] Example 1: Preparation of Compound I-1

[0060] In this example, 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).

[0061] Example 2: Preparation of Compounds II-1 to II-9

[0062] This example uses the experimental method described above to prepare compounds II-1 to II-9:

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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). 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,found 275.1325.

[0068] 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] + calcd 303.1643,found 303.1638.

[0069] 1 H 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.

[0070] 1 H NMR(400MHz,Chloroform-d)δ7.38(d,J=1.6Hz,1H),7.29(s,1H),6.59(d,J=8.2Hz,1 H),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.

[0071] 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). 13 C 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.

[0072] Example 3: Preparation of Compounds III-1 to III-15

[0073] This example uses the experimental method described above to prepare compounds III-1 to III-15:

[0074] 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, found 379.1951.

[0075] 1 H 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)δ169.73,163.06,150.06,148.19,139.86,135.54,122.61,1 21.62,119.90,111.77,111.18,51.64,47.75,33.40,25.88,24.94,15.71.HRMS(ESI)for C 22 H 25 N5S[M+H] + calcd 380.1909, found 380.1903.

[0076] 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.

[0077] 1 H 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.

[0078] 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). 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.

[0079] 1 H 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.

[0080] 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). 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.

[0081] 1 H 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,found 385.1515.

[0082] 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). 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.

[0083] 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.

[0084] 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). 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.

[0085] 1 H 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.

[0086] 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). 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.

[0087] 1 H 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). 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.

[0088] 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). 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.

[0089] Example 4: Preparation of Compounds IIII-1 to IIII-11

[0090] This example uses the experimental method described above to prepare compounds IIII-1 to IIII-11:

[0091] 1 H 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.

[0092] 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). 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.

[0093] 1 H 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.

[0094] 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). 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.

[0095] 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). 13 C 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.

[0096] 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.

[0097] 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). 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.

[0098] 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.

[0099] 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). 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.

[0100] 1 H 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.

[0101] 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),3.3 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.

[0102] Example 5: Preparation of Compound III-12

[0103] In this example, compound IIII-12 was prepared using the following experimental method:

[0104] 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.

[0105] (1) Synthesis of methyl 1,2,3,4-tetrahydroquinoline-6-carboxylate (Compound 2)

[0106] Methyl quinoline-6-carboxylate (0.3 g, 1.6 mmol) was added to a reaction flask, glacial acetic acid (20 mL) was used as solvent, and NaBH3CN (0.302 g, 0.48 mmol) was added in batches under ice bath conditions. The reaction was then carried out at room temperature until completion. The reaction solution was poured into a saturated aqueous NaHCO3 solution and extracted with ethyl acetate. The organic layers were combined and dried, and the mixture was concentrated in vacuo. The mixture was then separated by column chromatography to obtain a white solid (0.16 g, 52.28%).

[0107] 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).

[0108] (2) Synthesis of 1-benzyl-6-methyl 3-dihydroquinoline-1,6(2H)-dicarboxylate (Compound 3)

[0109] 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 20 mL of dichloromethane in a reaction flask. Cbz-Cl (0.67 g, 3.9 mmol) was added dropwise under an ice bath. The reaction was continued at room temperature until completion. The mixture was separated by column chromatography to obtain the title compound (0.39 g, 91.7%) as a white solid.

[0110] 1 H 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).

[0111] (3) Synthesis of 1-benzyl-6-methyl 8-nitro-3,4-dihydroquinoline-1,6(2H)-dicarboxylate (Compound 4)

[0112] Dissolve 1-benzyl-6-methyl 3-dihydroquinoline-1,6(2H)-dicarboxylate (0.3 g, 0.92 mmol) in a reaction flask containing trifluoroacetic acid (5 mL). Add NaNO2 (0.07 g, 1.01 mmol) in portions under ice-cooling. The mixture was allowed to react for 1 hour under ice-cooling and then for 2 hours at room temperature. After completion of the reaction, the mixture was poured into aqueous ammonia solution, the aqueous layer was extracted with EA, and the combined organic layers were dried. The mixture was concentrated in vacuo and then purified by column chromatography to yield a yellow oil (0.21 g, 61.76%).

[0113] 1H 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).

[0114] (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)

[0115] Benzyl 6-(2-acetylhydrazide-1-carbonyl)-8-nitro-3,4-dihydroquinoline-1(2H)-carboxylate (0.5 g, 1.21 mmol) and Lawesson's reagent (2.93 g, 7.26 mmol) were added to a reaction flask with dioxane (50 mL) as the solvent and refluxed overnight. The solvent was evaporated under vacuum, and the remaining mixture was dissolved in ethyl acetate. The organic layer was washed with saturated sodium bicarbonate solution, combined and concentrated, and the mixture was separated by column chromatography to obtain a yellow oil (0.25 g, 50.3%).

[0116] 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).

[0117] (5) Synthesis of Compound III-12

[0118] Compound 7 (0.2 g, 0.48 mmol) was added to a reaction flask containing 20 mL of methanol solution, followed by 10% palladium on carbon (0.04 g). The reaction was allowed to proceed under hydrogen for 12 h. After completion of the reaction, the palladium on carbon was filtered through celite, and the mixture was concentrated and purified by column chromatography to yield compound IIII-12 (0.07 g, 58.33%).

[0119] 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). 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.

[0120] Example 6: Ferroptosis Inhibitory Activity Test of Compounds

[0121] Studies have shown that GPX4 inhibitors such as RSL-3 can induce ferroptosis in cells, and this ferroptosis can also be blocked by other small molecules, such as lipophilic antioxidants such as 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.

[0122] Cell lines: 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.

[0123] Methods: MTT assay, as follows: Human renal carcinoma cell line OS-RC-2 and human neuroblastoma cell line SH-SY5Y in logarithmic growth phase were digested, harvested, and diluted. Approximately 4,000-5,000 cells were seeded per well of a 96-well plate. Three replicates were set up, with 80 μL per well. The cells were incubated overnight at 37°C in a 5% CO2 incubator. A DMSO control group and nine compound-treated groups with varying concentrations were included. Compounds were added to the treated groups at varying concentrations, and a DMSO control group (with the same dilution factor as the highest concentration compound) was established. After incubation in a 5% CO2, 37°C incubator for 1 hour, 5 μM RSL3 was added to each compound concentration to induce ferroptosis. RSL3 and DMSO control groups were also established and incubated in a 5% CO2, 37°C incubator for 48 hours. Add 20 μL of 5 mg / mL MTT solution to each well and continue incubating OS-RC-2 cells in a 37°C incubator for 2 hours. Then, add 100 μL of DMSO to each well and shake gently for 10 minutes to mix. Measure the optical density (OD) of each well at 570 nm using an enzyme-linked immunosorbent assay (ELISA). Repeat the experiment three times. Calculate the survival rate (%) using the following formula: OD value of the experimental group / OD value of the DMSO control group × 100%.

[0124] The results are shown in Table 1, which indicate that the various compounds of the present invention can significantly inhibit ferroptosis and have good ferroptosis inhibitory activity.

[0125] Table 1: Ferroptosis inhibition activity test results of compounds In the table: "A" indicates IC 50 ≤100nM, “B” indicates IC 50 >100nM and ≤500nM, “C” indicates IC 50 >500 nM and ≤1 μM, “D” indicates IC 50 >1μM and ≤10μM.

[0126] Example 7: In vitro / in vivo drug metabolism properties of compounds

[0127] In vitro testing methods: Plasma and liver microsomal homogenate samples were stored at -20°C, removed before use, and gradually warmed to room temperature. Compounds were dissolved in acetonitrile to prepare 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). At the end of the incubation period, the acetonitrile was terminated, the mixture was vortexed for 30 s, centrifuged at 12,000 rpm for 10 min, and then filtered through a 0.22 μM filter. The filtrate was analyzed using an HPLC system (Agilent 1260 HPLC) with an Alltima C18 column (5 μm, 4.6 mm × 250 mm). The flow rate was maintained at 400 μL / min, and the initial mobile conditions were 80% solvent A (0.1% acetic acid in water) and 20% solvent B (0.1% acetic acid in methanol). Solvent B was increased to 80% over 0.50 min and held for 1.50 min. Next, over 5.00 min, solvent B was increased to 100% and held there for 3 min. Next, solvent B was reduced to its initial condition (20%) over 0.50 min. The total run time was 12.00 min. The percentage of compound remaining at each time point was the ratio of the compound peak integral to the internal standard peak integral, where the internal standard peak area was the peak area at time t = 0.

[0128] The results are shown in Table 2 below. The Fer-1 structure-based thiadiazole compounds of the present invention exhibit good stability in plasma and liver microsomes in vitro, have excellent ADME properties, and can be used for in vivo biological activity studies.

[0129] Table 2: ADME property test results

[0130] In vivo testing: Following a single intravenous dose of the compound in male mice, blood and brain tissue samples were collected at 0, 0.5, 2, 4, and 8 hours. LC-MS / MS was used to determine the compound's concentration in mouse plasma and brain tissue, and relevant pharmacokinetic parameters were calculated to investigate the compound's pharmacokinetic characteristics and brain tissue distribution. The results indicate that compound I-1 exhibits good metabolic stability in mice, with a plasma half-life of 2.28 hours. It also crosses the blood-brain barrier, further demonstrating that this class of compounds can be used for in vivo studies and treatments of ferroptosis-related diseases.

[0131] Table 3: Pharmacokinetic parameters of I-1

[0132] Example 8: Compounds can be used as fluorescent probes to detect ferroptosis

[0133] Detection of ONOO by antioxidant fluorescent probe I-1 - Selectivity test: Under the same test conditions, excess amounts of other bioactive small molecules were added to the solution and the fluorescence spectrum was tested. The excitation wavelength was 350nm and the maximum emission wavelengths were 436nm and 525nm respectively. The results are shown in Figure 2. 1-14 represent the bioactive small molecules Blank, Cu 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 of the fluorescent probe prepared by the present invention to ONOO was significantly enhanced when other biologically active small molecules did not interfere with the detection results. - Has higher selectivity.

[0134] Detection of ONOO by antioxidant fluorescent probe I-1 - Fluorescence spectrum: In PBS buffer (pH = 7.4, 0.5% DMSO), a fluorescent probe with an initial concentration of 1mM was added to make the concentration of the fluorescent probe in the solution 10μM. Then, different amounts of ONOO with an initial concentration of 10mM were added in sequence. - , without adding ONOO - As a control, ONOO- Fully react with the fluorescent probe. Use fluorescence spectrometer to test different concentrations of ONOO - The fluorescence spectrum under the conditions of 350 nm excitation wavelength and 436 nm emission wavelength are shown in Figure 3. - With the increase of concentration, the fluorescence intensity at 436nm gradually increased, while the fluorescence intensity at 525nm gradually decreased.

[0135] Fluorescence spectra of the antioxidant fluorescent probe I-1 in different solvents: The fluorescent probe was added to different solvents at an initial concentration of 1 mM, resulting in a 20 μM concentration. Fluorescence spectra were then measured using 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, the maximum emission wavelength of I-1 gradually increased with increasing solvent polarity.

[0136] Flow cytometry was used to evaluate the antioxidant activity of I-1 and its self-indication ability: OS-RC2 cells were incubated with probe I-1, and the control group was incubated with antioxidant Fer-1. The cells were cultured in a 37°C incubator with 5% CO2 in a humidified environment. The cells were then incubated with RSL3 for 1 hour, and the RSL3 group was set as a reference control. Finally, BODIPY 581 / 591 After incubating the cells for 30 minutes, flow cytometry was performed, and untreated cells served as blank controls. As shown in Figure 5a, compared with the RSL3 group, the I-1-treated cells were able to effectively reverse the ferroptosis process, which was basically consistent with the results of the Fer-1 control group. In addition, the I-1 channel showed a significant fluorescence change (Figure 5b), indicating that the antioxidant fluorescent probe prepared by the present invention can effectively reverse the ferroptosis process and has self-indication ability.

[0137] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, characterized in that: The structural formula of the compound is shown below: In the formula, R 1 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-C 10 Aromatic heterocyclic group or C3-C7 saturated heterocyclic group; R 2 Selected from C0~C8 alkyl, C3~C 12 Cycloalkyl, adamantyl or polyalkynyl; R 3 is selected from hydrogen, alkyl, aryl, C1-C6 alkyl-aryl, C1-C6 alkyl-phenol or C3-C 10 Cycloalkyl; 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 group, C3-C7 saturated heterocyclic group, C3~C 12 Cycloalkyl, polyalkynyl, aryl, C1-C6 alkyl-aryl, C1-C6 alkyl-phenol or C3-C 10 Cycloalkyl groups can be substituted with one or more atoms or groups; n=0 or 2-4.

2. The compound according to claim 1, characterized in that The compounds are specifically compounds I-1, II-1 to II-9, III-1 to III-15, IIII-1 to IIII-12, and their structural formulas are as follows:

3. The method for preparing the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound according to claim 1, characterized in that: The preparation method comprises the following steps: (1) adding 4-chloro-3-nitrobenzoic acid, NH2NHBoc, HBTU and DIEPA to DMF for reaction, and separating and purifying the mixed solution to obtain a light yellow solid compound; (2) adding the compound of step (1) and Lawesson's reagent to dioxane, reacting at high temperature, and separating and purifying after completion to obtain a thiadiazole compound; (3) adding the thiadiazole compound and the corresponding amine of step (2) to DMSO for reaction, and separating and purifying the reaction mixture to obtain an intermediate compound; (4) adding the intermediate compound of step (3) and Pd / C to methanol, stirring and reacting under H2 conditions, and separating and purifying after completion to obtain an amino-containing compound; (5) Add the amino compound, the corresponding aldehyde and NaBH(OAc)3 of step (4) into DCM and stir to react. After the reaction, separate and purify to obtain a (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound.

4. The preparation method according to claim 3, characterized in that: In the step (1), the molar ratio of 4-chloro-3-nitrobenzoic acid, NH2NHBoc, HBTU and DIEPA is 1:1-2:1-2:1-2; and in the step (2), the molar ratio of the compound to Lawesson's reagent is 1:2-3.

5. The preparation method according to claim 3, characterized in that: In the step (3), the molar ratio of the thiadiazole compound to the amine is 1:1-2; in the step (4), the mass ratio of the intermediate compound to Pd / C is 10-12:1; and in the step (5), the molar ratio of the amino compound, the aldehyde and NaBH(OAc)3 is 1:1-1.5:2-3.

6. Use of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound according to claim 1 in the preparation of ferroptosis inhibitors.

7. Use of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound according to claim 1 in the preparation of drugs for ferroptosis-related diseases.

8. The use according to claim 7, characterized in that: The ferroptosis-related diseases include neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, liver and kidney failure, inflammation, and diabetic complications.

9. Use of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound according to claim 1 in the preparation of antioxidant fluorescent probes or antioxidant indicators.

10. The use according to claim 9, characterized in that: The antioxidant fluorescent probe or antioxidant indicator is used for ONOO - Highly sensitive detection, can effectively reverse the process of cell ferroptosis and has self-indication capabilities.

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