WO2025087033A1 - Compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine, preparation method therefor and use thereof - Google Patents

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 preparation method and application thereof Technical Field

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

Background Art

Cell death can be roughly divided into two categories: uncontrolled cell death caused by excessive cell damage and regulated cell death that relies on strictly controlled molecular pathways. Apoptosis is the most typical form of regulated cell death, which triggers cell death by activating caspases. Ferroptosis is a new type of cell death discovered in recent years. It is an oxidative cell death induced by various reasons and is iron-dependent. Its occurrence is caused by the imbalance of the generation and degradation of reactive oxygen species (ROS) in intracellular lipids. The control mechanism of ferroptosis elucidated in the first few years of the discovery of ferroptosis mainly revolves around cysteine and glutathione metabolism, and phospholipid peroxidase GPX4 prevents the accumulation of peroxidized lipids. Ferroptosis inducers act directly or indirectly on glutathione peroxidase (GPXs) through different pathways, resulting in reduced cellular antioxidant capacity, ROS accumulation, and ultimately cell oxidative death. The complex interaction between lipid, iron and cysteine metabolism has become an important regulator of this cell death pathway. Recently, modulation of ferroptosis has emerged as an attractive strategy for intervention in human diseases, including cancer, neurodegenerative diseases, and ischemic disorders.

Ferroptosis can be inhibited by iron chelators, lipophilic antioxidants, and/or ferrostatin-1 (fer-1). Fer-1 is an arylalkylamine with antioxidant properties and was one of the first identified ferroptosis inhibitors. Fer-1 acts as a lipid peroxidation reductant, intercepting and scavenging lipid free radicals via hydrogen atom transfer or direct reduction. However, its short half-life makes it unsuitable for further pharmacological evaluation.

In recent years, high-sensitivity and 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 scientific research activities related to ferroptosis, fluorescent probes are also 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 for ^ONOO- recognition in organisms, such as rhodamine and Nile red compounds. However, these small molecules only have recognition functions and are not suitable for use as antioxidant drugs. Therefore, they still have certain limitations in practical applications.

Normally, fluorescent probes detect substances by increasing or decreasing fluorescence intensity. Based on this, a fluorescent probe that can react with ^ONOO- is designed. This probe can consume ^ONOO- through chemical reactions to play an antioxidant role. At the same time, the changes in the fluorescence spectrum caused before and after the reaction enable it to have real-time monitoring functions. This type of antioxidant fluorescent probe with self-indicating function can achieve self-monitoring in the process of reversing ferroptosis without the need for additional processing of experimental samples, which provides a new idea for the development of evaluation methods for ferroptosis antioxidants.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine and a preparation method and application thereof. The compound has strong antioxidant activity and good metabolic stability, and is suitable for the treatment of ferroptosis-related diseases; and has obvious fluorescence spectrum changes, has extremely strong selectivity for ONOO ^- , and the phenomenon is obvious and easy to identify.

The objective of the present invention is achieved through the following technical solutions:

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:

In the formula, ^R1 is selected from hydrogen, _C1 - _C6 alkyl, _C1 - _C6 alkoxy, C2- _C6 alkenyl, _C2 - _C6 alkynyl, _C3 - _C8 cycloalkyl, _C3 - _C8 cycloalkoxy, amino, phenyl, benzyl, naphthyl, _C5 - _C10 aromatic heterocyclic group or _C3 - _C7 saturated heterocyclic group _;

R ² is selected from C ₀ ～C ₈ alkyl, C ₃ ～C ₁₂ cycloalkyl, adamantyl or polyalkynyl;

R ³ is selected from hydrogen, alkyl, aryl, C ₁ -C ₆ alkyl-aryl, C ₁ -C ₆ alkyl-phenol or C ₃ ～C ₁₀ cycloalkyl;

Wherein, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ cycloalkyloxy, phenyl, benzyl, naphthyl, C ₅ -C ₁₀ aromatic heterocyclic group, C ₃ -C ₇ saturated heterocyclic group, C ₃ ～C ₁₂ cycloalkyl, polyalkynyl, aryl, C ₁ -C ₆ alkyl-aryl, C ₁ -C ₆ alkyl-phenol group or C ₃ ～C ₁₀ cycloalkyl group can be substituted by one or more atoms or groups;

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

Preferably, R ¹ is selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ cycloalkoxy, aryl and derivatives.

Preferably, R ² is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and derivatives thereof, cycloheptyl, adamantyl and derivatives thereof.

Preferably, R ³ is selected from cyclopropyl, cyclopentyl, cyclohexyl, arylbenzyl and derivatives thereof.

Preferably, n=0, 2-4.

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:

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:

(1) Adding compound 4-chloro-3-nitrobenzoic acid, NH ₂ NHBoc, HBTU and DIEPA into DMF, reacting at room temperature for 4 hours, and separating and purifying the mixed solution after the reaction to obtain a light yellow solid compound 2;

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

(3) adding the thiadiazole compound 3 prepared in step (2) and the corresponding amine to DMSO, reacting at 60° C. for 18 hours, and separating and purifying after the reaction to obtain the corresponding intermediate compound 4;

(4) adding the intermediate compound 4 of step (3) and Pd/C to methanol, stirring at room temperature for 6 hours under _H2 conditions, reacting until completion, and then separating and purifying to obtain an amino-containing compound 5;

(5) Add the amino compound 5 of step (4), the corresponding aldehyde and NaBH(OAc) ₃ into DCM, stir at room temperature for 6 hours, and after the reaction is completed, separate and purify to obtain a (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound 6.

Preferably, in step (1), the molar ratio of 4-chloro-3-nitrobenzoic acid, NH ₂ NHBoc, HBTU and DIEPA is 1:1.5:1.5:1.5.

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

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

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

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

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.

Preferably, the specific method of separation and purification in step (2) is: after the reaction is completed, cool to room temperature, evaporate the solvent, extract the mixture with water and ethyl acetate, combine the organic layers, and then wash the combined organic layers with 10% sodium bicarbonate solution. Then, dry the organic layer with sodium sulfate, filter, concentrate and separate by column chromatography to obtain thiadiazole compound 3.

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.

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;

Preferably, the specific method of separation and purification in step (5) is: after the reaction is completed _, the reaction solution is poured into a saturated sodium bicarbonate aqueous solution, the aqueous layer is extracted with _CH2Cl2 , the combined organic layer is washed with a saturated sodium bicarbonate aqueous solution, and then washed with brine, dried, filtered, and concentrated in vacuo. The mixture is separated by column chromatography to obtain a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine.

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

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

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

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.

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.

Furthermore, the antioxidant fluorescent probe or antioxidant indicator is used for high-sensitivity detection of ONOO- ^, which can effectively reverse the process of cell ferroptosis and has self-indication capability.

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

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;

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

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. In the process of inhibiting ferroptosis, the effect of the monitoring effect can be evaluated in real time through the changes in its own fluorescence spectrum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1A is a hydrogen nuclear magnetic resonance spectrum of compound I-1; FIG1B is a carbon spectrum of compound I-1; and FIG1C is a high-resolution mass spectrum of compound I-1;

Fig. 2 is a graph showing the selectivity test results of the antioxidant fluorescent probe I-1 for detecting ONOO ^- , wherein 1-14 represent the bioactive small molecules Blank, Cu ²⁺ , Zn ²⁺ , Fe ³⁺ , Fe ²⁺ , Al ³⁺ , Ca ²⁺ , SO ₄ ^2- , SO ₃ ^2- , NO ₂ ^- , O ^2- , t-BuOOH, H ₂ O ₂ , HClO, ONOO ^- , ·OH, Vc ^- , GSH, and VC, respectively;

FIG3 is a fluorescence spectrum result diagram of the antioxidant fluorescent probe I-1 detecting ONOO ^- ;

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

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

DETAILED DESCRIPTION

The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

The general synthesis route of a compound containing (1,3,4-thiadiazole)benzene-1,2-ethylenediamine is as follows:

Wherein: (a) HBTU, DIEPA, NH ₂ NHCOR ¹ ; (b) Lawes' reagent, reflux; (c) corresponding amine, K ₂ CO ₃ , DMSO, 80° C.; (d) Pd/C, H ₂ , CH ₃ OH; (e) corresponding aldehyde, NaBH(OAc) ₃ , DCM.

(1) Synthesis method a: Compound 1 (4.96 mmol), NH ₂ NHCOR ¹ (7.44 mmol), HBTU (7.44 mmol) and DIEPA (7.44 mmol) were added to a reaction bottle containing DMF (3 mL) and reacted at room temperature until completion. 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 light yellow solid 2.

(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 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 with sodium sulfate, filtered, concentrated and separated by column chromatography to obtain a yellow solid, which was thiadiazole compound 3.

(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, DMSO (3 mL) was used as solvent, and the reaction solution was reacted at 60°C until the reaction was completed as 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.

(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 celite pad and the solvent was removed under reduced pressure. The residue was purified by silica gel flash column chromatography to obtain the desired compound 5.

(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 sodium bicarbonate aqueous solution and the aqueous layer was extracted with CH ₂ Cl _2. The combined organic layers were washed with a saturated sodium bicarbonate aqueous solution and then with brine, dried, filtered, and concentrated in vacuo. The mixture was separated by column chromatography to obtain the target compound 6.

Example 1: Preparation of Compound I-1

In this example, compound I-1 was prepared by the experimental method described above. ¹ H NMR (400 MHz, Chloroform-d) δ7.39 (s, 1H), 7.29 (d, J = 7.1 Hz, 1H), 6.62 (d, J = 8.3 Hz, 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 N ₄ S[M+H] ⁺ calcd 289.1487, found 289.1481 (Figure 1).

Example 2: Preparation of Compounds II-1 to II-9

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

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-d ₆ )δ169.82,162.79,140.46,135.41,118.98,118.18,112.14,108.87,30.30,15.65.

¹ H 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.

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.

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.

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). ^13C NMR(101MHz,Chloroform-d)δ169.66,163.00,140.81,133.49,122.01,119.42,1 15.36,119.42,115.36,111.25,54.42,33.63,29.71,24.26,15.71.HRMS(ESI)for C ₁₄ H ₁₈ N ₄ S[M+H] ⁺ calcd 275.1330, found 275.1325.

^{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 N ₄ S[M+H] ⁺ calcd 303.1643, found 303.1638.

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). ¹³ C NMR (101MHz, Chloroform-d) δ 169.23, 163.48, 141.96, 136.13, 132.28, 125.65, 120.86, 115.92, 42.48, 36.32, 29.64, 22.70, 15.74.

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) δ 169.73, 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.

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 N ₅ O ₂ S[M+H] ⁺ calcd 390.1964, found 390.1958.

Example 3: Preparation of Compounds III-1 to III-15

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

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 N ₄ S[M+H] ⁺ calcd 379.1956, found 379.1951.

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). ^13C 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 N ₅ S[M+H] ⁺ calcd 380.1909,found 380.1903.

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＝8.5,2.0 Hz,1H),6.69(d,J＝8.6Hz,1H),3.45–3.35(m,1H),2.77(s,3H),2.13–2.05(m,2H),1.80(m,2H),1.67(m,2H),1.48–1.31(m,4H). ^13C NMR(101MHz,Chloroform-d)δ169.26,162.94,155.25,150.51,145.53,142.84,13 4.78,129.66,122.23,117.63,115.54,110.52,51.17,33.09,25.76,24.82,15.74.

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,2 H),3.28(m,1H),2.75(s,3H),2.04(t,J＝4.7Hz,2H),1.80–1.72(m,2H),1.68–1.61(m,1H),1.38(dd,J＝16.5,8.3Hz,5H). ^13C NMR(101MHz, Methanol-d ₄ )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.

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). ^13C NMR(101MHz,Chloroform-d)δ169.92,162.97,139.77,136.02,129.61,121.32,115.62, 115.41,111.73,110.81,51.65,48.44,33.38,29.70,25.88,24.97,15.71.HRMS(ESI)for C ₂₂ H ₂₅ FN ₄ S[M+H] ⁺ calcd 397.1862,found 397.1857.

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). ^13C NMR(101MHz,Chloroform-d)δ170.03,162.88,139.70,137.16,136.30,135.94,129.3 5,128.18,121.05,111.52,110.57,51.62,48.94,33.36,25.89,25.00,21.14,15.69.

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). ^13C NMR(101MHz,Chloroform-d)δ169.85,163.00,148.51,141.31,139.97,135.44,133.98,129.5 4,122.61,122.43,121.68,119.62,111.57,111.02,51.69,48.13,33.33,25.89,24.98,15.69.

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),4.3 5(s,2H),3.30(m,1H),2.75(s,3H),2.10–2.03(m,2H),1.81–1.74(m,2H),1.71–1.51(m,3H),1.45–1.29(m,3H). ^13C NMR(101MHz,Chloroform-d)δ169.93,162.98,145.52,139.94,129.26,127.58,126. 18,122.28,121.61,111.89,44.37,33.26,29.70,25.87,24.97,15.72.HRMS(ESI)for C ₂₀ H ₂₄ N ₄ S ₂ [M+H] ⁺ calcd 385.1521, found 385.1515.

^1H NMR(400MHz,Chloroform-d)δ8.14(d,J＝8.5Hz,1H),8.09(d,J＝8.6Hz,1H),7.82 (d,J＝8.4Hz,1H),7.77–7.70(m,1H),7.53(t,J＝7.9Hz,1H),7.46–7.41(m,2H),7 .29–7.26(m,1H),6.69(d,J＝8.1Hz,1H),4.66(s,2H),3.38(m1H),2.74(s,3H),2 .13(dd,J＝12.5,3.7Hz,2H),1.82(m,2H),1.71–1.63(m,1H),1.47–1.31(m,5H). ^13C NMR(101MHz,Chloroform-d)δ169.65,164.33,151.96,150.30,147.20,143.51,136.79,129.9 8,127.76,127.58,122.56,122.25,120.83,114.03,57.55,51.58,31.49,29.71,26.31,15.78.

^1H NMR(400MHz,Chloroform-d)δ7.36(s,1H),7.23(s,1H),6.65(d,J＝8.2Hz,1H),3.80(p,J＝6.2Hz,1H),3.3 1–3.19(m,1H),2.75(s,3H),2.38–2.30(m,2H),2.05(m,4H),1.75(m,5H),1.67–1.58(m,5H),1.52(m,2H). ^13C NMR(101MHz,Chloroform-d)δ160.76,144.91,122.30,121.81,119.12,111. 34,108.52,49.39,42.83,29.27,28.02,27.49,23.76,22.79,21.00,13.52.

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). ^13C NMR(101MHz,Chloroform-d)δ162.87,136.05,120.71,119.71,112.45,110. 72,108.28,55.07,51.63,33.59,33.43,29.70,25.93,24.98,24.32,15.70.

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). ^13C NMR(101MHz,Chloroform-d)δ165.54,163.03,141.19,124.47,123.98,5119.11,112.99,111.4 2,54.53,52.52,41.99,35.69,33.54,31.94,31.44,29.37,25.99,25.00,24.29,22.70,14.13.

^1H NMR (400MHz, Chloroform-d) δ7.34(s,1H),7.22(d,J＝6.8Hz,1H),6.63(d,J＝8.1Hz,1H),3.53(d,J＝5.1Hz,1H),3.27(s,1H),2.73( s,3H),2.06(t,J＝14.2Hz,3H),1.75(d,J＝12.3Hz,4H),1.65(m,3H),1.54(m,2H),1.42–1.26(m,7H),0.91(dd,J＝11.8,6.4Hz,3H). ^13C NMR(101MHz,Chloroform-d)δ170.10,162.89,140.65,135.45,120.89,119.80,113.31 ,111.46,65.23,51.54,41.98,34.00,33.36,32.42,29.95,25.93,24.84,22.25,15.67.

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,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). ^13C NMR(101MHz,Chloroform-d)δ162.88,140.81,135.06,121.23,113.60,110.96,52.52,51.53,41.98,33.60,29.66,26.02,24.99,15.68.

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). ^13C NMR(101MHz,Chloroform-d)δ170.08,162.28,142.10,138.80,122.12,117.76,104.78,52.85,45.46,30.25,29.70,26.21,25.52,15.68.

Example 4: Preparation of Compounds IIII-1 to IIII-11

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

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). ^13C NMR(101MHz,Chloroform-d)δ169.91,160.41,139.67,133.49,121.93,119.49,115.89,111.17,51.71,33.22,25.86,24.94,23.85,14.40.

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, CDCl ₃ )δ169.14,168.40,139.76,133.45,122.01,119.55,115.93,111.17,51.68,33.26,32.11,25.87,24.95,23.47,13.64.

^1H NMR(400MHz,Chloroform-d)δ7.37(d,J＝2.0Hz,1H),7.27(d,J＝2.1Hz,1H),7.25(s,1H),6.63(d,J＝8.3Hz,1H),3.31(m,1 H),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, CDCl ₃ )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.

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.

^1H NMR(400MHz,Chloroform-d)δ8.00–7.96(m,2H),7.50–7.45(m,4H),7.40(dd,J=8.3,2.1Hz,1H),6.66(d,J=8. 4Hz,1H),3.33(m,1H),2.14–2.04(m,2H),1.80(m,2H),1.68(m,2H),1.44–1.35(m,2H),1.30(q,J＝3.2Hz,2H). ¹³ C NMR (400MHz, CDCl ₃ )δ168.80,166.40,133.41,130.68,130.54,129.09,127.76,121.20,116.26,113.06,50.85,33.09,29.71,25.82,24.93.

^11H NMR(400MHz,Chloroform-d)δ7.83(d,J＝8.6Hz,2H),7.59(d,J＝8.6Hz,2H),7.46(d,J＝1.9Hz,1H),7.38(dd,J＝8.3,1.9Hz,1 H),6.65(d,J＝8.4Hz,1H),3.33(m,1H),2.13–2.03(m,2H),1.79(m,2H),1.68(m,2H),1.46–1.33(m,2H),1.31–1.22(m,4H). ^13C NMR(101MHz,Chloroform-d)δ165.17,132.29,129.06,124.99,122.28,116.13,51.89,33.12,29.71,25.82,24.92.

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, CDCl ₃ )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.

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). ^13C NMR(101MHz,Chloroform-d)δ180.18,151.32,148.62,134.59,123.93,116.27,52.22,33.08,29.70,25.77,24.91.

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). ^13C NMR(101MHz,Chloroform-d)δ173.10,154.99,154.61,154.22,153.83,141.37,138.68,135.96 ,128.75,128.12,127.61,122.22,117.47,112.07,110.25,51.58,49.18,33.29,25.82,24.96.

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). ^13C NMR(101MHz,Chloroform-d)δ173.16,154.96,154.58,154.19,153.80,142.34,134.80,122. 30,117.27,114.10,110.48,52.58,51.45,33.60,33.25,29.72,25.99,25.87,24.97,24.89.

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). ^13C 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.

Example 5: Preparation of Compound IIII-12

This example uses the following experimental method to prepare compound IIII-12:

Among them, (a) NaBH ₃ CN, CH ₃ COOH; (B) CbzCl, TEA, DCM; (c) CF ₃ COOH, NaNO ₂ ; (d) LiOH, THF; (e) HBTU, DIEPA, NH ₂ NH ₂ Ac; (f) Lawesson's Reagent, THF, reflux; (g) H ₂ , Pd/C, CH ₃ OH.

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

Quinoline-6-carboxylic acid methyl ester (0.3 g, 1.6 mmol) was added to a reaction flask, glacial acetic acid (20 mL) was used as solvent, NaBH ₃ CN (0.302 g, 0.48 mmol) was added in batches under ice bath conditions, and then the reaction was completed at room temperature. The reaction solution was poured into a saturated NaHCO ₃ aqueous solution, extracted with ethyl acetate, the organic layers were combined and dried, and the mixed solution was concentrated in vacuo, and then separated by column chromatography to obtain a white solid (0.16 g, 52.28%).

¹ H NMR (400MHz, DMSO-d ₆ )δ7.48–7.43(m,2H),6.61(s,1H),6.42(d,J＝8.2Hz,1H),3.71(s,3H),3.22(m,2H),2.67(t,J＝6.2Hz,2H),1.80–1.73(m,2H).

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

Dissolve 1,2,3,4-tetrahydroquinoline-6-carboxylic acid methyl ester (0.25 g, 1.3 mmol) and DIEPA (0.67 g, 5.2 mmol) in a reaction bottle containing 20 mL of dichloromethane, add Cbz-Cl (0.67 g, 3.9 mmol) dropwise under ice bath conditions, and then react 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.

¹ H NMR (400MHz, DMSO-d ₆ )δ7.86(d,J＝8.4Hz,1H),7.71(d,J＝8.3Hz,2H),7.44–7.31(m,5H),5.21(s,2H) ,3.81(s,3H),3.79–3.73(m,2H),2.78(t,J＝6.4Hz,2H),1.86(p,J＝6.3Hz,2H).

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

3-Dihydroquinoline-1,6(2H)-dicarboxylic acid 1-benzyl-6-methyl (0.3 g, 0.92 mmol) was dissolved in a reaction bottle containing trifluoroacetic acid (5 mL), and NaNO ₂ (0.07 g, 1.01 mmol) was added in batches under ice bath conditions, and the reaction was carried out under ice bath conditions for 1 h, and then at room temperature for 2 h. After the reaction was completed, the mixed solution was poured into an aqueous ammonia solution, the aqueous layer was extracted with EA, the organic layers were combined and dried, and the mixed solution was concentrated in vacuo, and then separated by column chromatography to obtain a yellow oil (0.21 g, 61.76%).

¹ H NMR (400MHz, DMSO-d ₆ )δ8.21(d,J＝1.9Hz,1H),8.07(d,J＝2.0Hz,1H),7.47–7.19(m,5H),5.31–5.02(m,2H),3.88(s,5H),3.05–2.72(m,2H),2.11–1.65(m,2H).

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

Benzyl 6-(2-acetylhydrazine-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, dioxane was used as solvent (50 mL), and the reaction was refluxed overnight. The solvent was evaporated under vacuum conditions, and the remaining mixture was dissolved in ethyl acetate, and the organic layer was washed with a saturated sodium bicarbonate solution. The organic layers were then combined and concentrated, and the mixture was separated by column chromatography to obtain a yellow oil (0.25 g, 50.3%).

¹ H NMR (400MHz, DMSO-d ₆ )δ8.23(s,1H),8.06(s,1H),7.39–7.25(m,5H),5.16(s,2H),3.99–3.66(m,2H),2.89(s,2H),2.76(s,3H),2.00–1.81(m,2H).

(5) Synthesis of Compound IIII-12

Compound 7 (0.2 g, 0.48 mmol) was added to a reaction bottle containing 20 mL of methanol solution, and 10% palladium carbon (0.04 g) was added, and the reaction was carried out under hydrogen for 12 hours. After the reaction was completed, the palladium carbon was filtered off with diatomaceous earth, and the mixed solution was concentrated and subjected to column chromatography to obtain compound IIII-12 (0.07 g, 58.33%).

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.

Example 6: Ferroptosis inhibition activity test of compounds

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

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.

Methods: MTT method, as follows: Human renal cancer cell line OS-RC-2/human neuroblastoma cell SH-SY5Y in logarithmic growth phase were digested, collected and diluted, and about 4000-5000 cells were seeded in each well of 96-well plate. The experiment set up 3 replicate wells, 80μL per well. Incubate overnight in a 37℃, 5% CO ₂ incubator. The experiment set up a DMSO control group and nine different concentrations of compound administration groups. Different concentrations of compounds were added to the administration group, and a DMSO control group (the same dilution multiple as the highest concentration compound) was set up. After incubation in a 5% CO ₂ , 37℃ incubator for 1h, each compound concentration was added with 5μM RSL3 to induce ferroptosis, and RSL3 control group and DMSO control group were set up. Incubation continued for 48h in a 5% CO ₂ , 37℃ incubator. Add 20 μL of 5 mg/mL MTT solution to each well, continue to culture OS-RC-2 cells in a 37°C incubator for 2 hours, add 100 μL of DMSO to each well, shake steadily for 10 minutes, and mix. Place on an enzyme-linked immunosorbent assay detector, and measure the optical density value (OD value) of each well at a wavelength of 570 nm. Repeat the experiment 3 times. Calculate according to the following formula: Survival rate % = OD value of experimental group/OD value of DMSO control group × 100%.

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.

Table 1: Test results of ferroptosis inhibition activity of compounds



In the table: "A" indicates IC ₅₀ ≤100nM, "B" indicates IC ₅₀ >100nM and ≤500nM, and "C" indicates IC ₅₀ >500
nM and ≤1 μM, "D" indicates _IC50 >1 μM and ≤10 μM.

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

In vitro test method: Plasma and liver microsomal homogenate samples were stored at -20 °C, taken out before use, and gradually warmed to room temperature. The compound was dissolved in acetonitrile to make a 4 mg/mL stock solution, then added to plasma or liver microsomal homogenate to a final concentration of 0.4 mg/mL and incubated at 37 °C for the desired time (0, 0.25, 0.5, 1, 2, 4, 8, 12, 24 and 24 h). The acetonitrile was terminated at the end point of the incubation, the mixture was vortexed for 30 s and centrifuged at 12000 rpm for 10 min, and then filtered through a 0.22 μM filter. The filtrate was analyzed by HPLC system (Agilent 1260 HPLC) with Alltima C18 (5 μm, 4.6 mm × 250 mm) column. The flow rate was maintained at 400 μL/min, and the initial flow conditions were 80% solvent A (water containing 0.1% acetic acid) and 20% solvent B (methanol containing 0.1% acetic acid). Solvent B was increased to 80% in 0.50 min and maintained for 1.50 min. Then, solvent B was increased to 100% in 5.00 min and maintained there for 3 min. Then, solvent B was reduced to initial conditions (20%) in 0.50 min. The total running time was 12.00 min. The percentage of compound remaining at each time point = the ratio of the compound peak integral to the internal standard peak integral, and the internal standard peak area was the peak area at t = 0 time point.

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 good ADME properties, and can be used for in vivo biological activity studies.

Table 2: ADME property test results

In vivo test method: After a single dose of intravenous injection of the compound in male mice, blood samples and brain tissue were collected at 0, 0.5h, 2h, 4h, and 8h. The concentration of the compound in mouse plasma and brain tissue was determined by LC-MS/MS and the relevant pharmacokinetic parameters were calculated to investigate the pharmacokinetic characteristics and brain tissue distribution of the compound in mice. From the results, it can be seen that compound I-1 exhibits good metabolic stability in mice, with a half-life of 2.28h in plasma, and it can pass through the blood-brain barrier, further indicating that such compounds can be used for in vivo research and treatment of diseases related to ferroptosis.

Table 3: Pharmacokinetic parameters of I-1

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

Selectivity test of antioxidant fluorescent probe I-1 for detecting ONOO ^- : Under the same test conditions, an excess of other bioactive small molecules were added to the solution, and the fluorescence spectrum was tested, with an excitation wavelength of 350 nm and maximum emission wavelengths of 436 nm and 525 nm, respectively. The results are shown in FIG2 , where 1-14 represent bioactive small molecules Blank, Cu ²⁺ , Zn ²⁺ , Fe ³⁺ , Fe ²⁺ , Al ³⁺ , Ca ²⁺ , SO ₄ ^2- , SO ₃ 2- , NO ^2- , O _2- , t ^- BuOOH, H ₂ O ₂ , HClO, ONOO ^- , ·OH, Vc ^- , GSH, VC. ^F ₄₃₆ /F ₅₂₅ is significantly enhanced only when ONOO ^- exists, and other bioactive small molecules do not interfere with the test results, indicating that the fluorescent probe prepared by the present invention has a high selectivity for ONOO ^- .

Antioxidant fluorescent probe I-1 detects fluorescence spectrum of ONOO ^- : In PBS buffer (pH = 7.4, 0.5% DMSO), add the fluorescent probe with an initial concentration of 1mM to make the concentration of the fluorescent probe in the solution 10μM. Then, add different amounts of ONOO ^- with an initial concentration of 10mM in sequence, and do not add ONOO ^- as a control, and allow ONOO ^- to fully react with the fluorescent probe at 37℃ for 90min. The fluorescence spectrum under different concentrations of ONOO ^- was tested with a fluorescence spectrometer. The excitation wavelength of the fluorescence spectrum was 350nm, and the emission wavelengths were 436nm and 525nm. As shown in Figure 3, as the concentration of ONOO ^- increases, the fluorescence intensity at 436nm gradually increases, and the fluorescence intensity at 525nm gradually decreases.

Fluorescence spectra of antioxidant fluorescent probe I-1 in different solvents: In different solvents, the fluorescent probe was added with an initial concentration of 1 mM to make the concentration of the fluorescent probe in the solution 20 μM. Then, the fluorescence spectra in different solvents were tested with a fluorescence spectrometer. The excitation wavelength of the fluorescence spectrum was 350 nm and the emission wavelength was 525 nm. As shown in Figure 4, with the increase of solvent polarity, the maximum emission wavelength of I-1 gradually increased.

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 an incubator at 37°C in a humid environment with 5% _CO2 . The above cells were then incubated with RSL3 for 1 hour, and the RSL3 group was set as a reference control. Finally, the cells were incubated with BODIPY ^581/591 for 30 minutes and then subjected to flow cytometry testing. Cells without any treatment were used as blank controls. As can be seen from Figure 5a, compared with the cells in the RSL3 group, the cells treated with I-1 can effectively reverse the process of cell ferroptosis, which is basically consistent with the results of the Fer-1 control group. In addition, the I-1 channel showed obvious fluorescence changes (Figure 5b), indicating that the antioxidant fluorescent probe prepared by the present invention can effectively reverse the process of cell ferroptosis and has self-indication ability.

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 by 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 (10)

A (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound, characterized in that the structural formula of the compound is as follows:
In the formula, ^R1 is selected from hydrogen, _C1 - _C6 alkyl, _C1 - _C6 alkoxy, C2- _C6 alkenyl, _C2 - _C6 alkynyl, _C3 - _C8 cycloalkyl, _C3 - _C8 cycloalkoxy, amino, phenyl, benzyl, naphthyl, _C5 - _C10 aromatic heterocyclic group or _C3 - _C7 saturated heterocyclic group _; R ² is selected from C ₀ ～C ₈ alkyl, C ₃ ～C ₁₂ cycloalkyl, adamantyl or polyalkynyl; R ³ is selected from hydrogen, alkyl, aryl, C ₁ -C ₆ alkyl-aryl, C ₁ -C ₆ alkyl-phenol or C ₃ ～C ₁₀ cycloalkyl; Wherein, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ cycloalkyloxy, phenyl, benzyl, naphthyl, C ₅ -C ₁₀ aromatic heterocyclic group, C ₃ -C ₇ saturated heterocyclic group, C ₃ ～C ₁₂ cycloalkyl, polyalkynyl, aryl, C ₁ -C ₆ alkyl-aryl, C ₁ -C ₆ alkyl-phenol group or C ₃ ～C ₁₀ cycloalkyl group can be substituted by one or more atoms or groups; n=0 or 2-4. The compound according to claim 1, characterized in that the compound is specifically compound I-1, II-1 to II-9, III-1 to III-15, IIII-1 to IIII-12, and its structural formula is as follows:

The method for preparing the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound according to claim 1, characterized in that the method comprises the following steps: (1) adding 4-chloro-3-nitrobenzoic acid, NH ₂ NHBoc, HBTU and DIEPA to DMF for reaction, and after the reaction, separating and purifying the mixture 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 is completed, separate and purify to obtain a (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound. The preparation method according to claim 3, characterized in that the molar ratio of 4-chloro-3-nitrobenzoic acid, _NH2NHBoc , HBTU and DIEPA in step (1) is 1:1-2:1-2:1-2; and the molar ratio of the compound and Lawesson's reagent in step (2) is 1:2-3. The preparation method according to claim 3 is characterized in that the molar ratio of the thiadiazole compound to the amine in step (3) is 1:1-2; the mass ratio of the intermediate compound to Pd/C in step (4) is 10-12:1; and the molar ratio of the amino compound, aldehyde and NaBH(OAc) ₃ in step (5) is 1:1-1.5:2-3. Use of the (1,3,4-thiadiazole)benzene-1,2-ethylenediamine compound according to claim 1 in the preparation of ferroptosis inhibitors. The 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. The use according to claim 7 is characterized in that the ferroptosis-related diseases include neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular, liver and kidney failure, inflammation, and diabetic complications. 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. The use according to claim 9 is characterized in that the antioxidant fluorescent probe or antioxidant indicator is used for high-sensitivity detection of ^ONOO- , can effectively reverse the process of cell ferroptosis and has self-indication ability.