CN116199680B - Endoperoxide compound containing GPX4 protein covalent group, and preparation method and application thereof - Google Patents

Abstract

The invention provides an endoperoxide compound containing GPX4 protein covalent groups, and a preparation method and application thereof, belonging to the field of chemical medicine. The endoperoxide compound containing the GPX4 protein covalent group has a certain proliferation inhibition effect on MCF-7 cells (human breast cancer cells) in-vitro antitumor activity evaluation, and can be used as a potential antitumor compound for developing antitumor drugs.

Description

Endoperoxide compounds containing covalent groups of GPX4 protein and preparation methods and applications thereof

Technical field

The invention relates to a type of endoperoxide compound containing a GPX4 protein covalent group and its preparation method and application, and belongs to the field of chemical medicine.

Background technique

Ferroptosis was proposed in 2012. It is a new type of cell death that is dependent on iron ions and characterized by the accumulation of intracellular reactive oxygen species and lipid peroxidation. Its biochemical characteristics include increased lipid peroxides and iron ion accumulation. Its ultrastructural morphological characteristics show cell membrane rupture, smaller mitochondria and increased double membrane density, reduced or disappeared mitochondrial cristae, ruptured outer membrane, normal nucleus size but lack of chromatin condensation, etc. Since the discovery of this process, many small molecule inducers of this pathway have been discovered, such as erastin, FINO ₂ , ML210, etc.

Both FINO ₂ and artemisinin derivatives can induce ferroptosis in different types of tumor cells. The common structural feature of both is that they both contain peroxygen bonds, which can react with Fe ²⁺ to produce ROS in a Fenton-like reaction. This in turn induces lipid peroxidation and triggers ferroptosis. Therefore, the peroxygen bond is the key pharmacophore for these two types of compounds. In addition, iron ions participate in various physiological processes such as DNA synthesis in the body and are an indispensable element for cell survival. Due to abnormally active division, tumor cells have an increased demand for iron compared with normal cells. There are more transferrin receptors on the cell surface, so the intracellular iron content is higher than that of normal cells. This is due to FINO ₂ and artemisinins. One of the reasons why derivatives have specific inhibitory effects on tumor cells.

GPX4 is a key antioxidant enzyme that reduces toxic lipid peroxides and regulates ferroptosis. Inhibiting GPX4 activity or knocking out GPX4 can inhibit tumor growth. Studies have shown that in a treatment-induced drug-resistant state, the levels of polyunsaturated lipids in cancer cells increase, thereby increasing the cancer cell's dependence on GPX4 to prevent ferroptosis caused by lipid peroxidation. GPX4 is a difficult drug target because the surface of the selenocysteine in the catalytic site of the enzyme is relatively flat and lacks possible drug-binding pockets. This structural feature makes GPX4 less likely to be targeted by small molecule inhibitors. . Existing GPX4 inhibitors are all covalent inhibitors, which covalently bind to the selenocysteine residue in the catalytic center of GPX4 through an electrophilic warhead. But so far, there is still a lack of highly efficient and low-toxic GPX4 inhibitors.

Therefore, it is considered to introduce a targeted electrophilic warhead of GPX4 into the endoperoxide structure, and use the principle of splicing to design and synthesize a series of endoperoxide compounds containing GPX4 covalent inhibitory groups. Compared with drug combination therapy, single molecules are more Mechanistic therapy can reduce drug-drug interactions and adverse reactions, and more effectively improve anti-tumor efficacy.

Contents of the invention

The endoperoxide compounds containing GPX4 protein covalent groups according to the present invention can induce ferroptosis in tumor cells and have the following structural formulas 1b, 2b, 3b and 4b:

The preparation method of the endoperoxide compound containing the covalent group of the GPX4 protein includes the following steps: ① The raw material methyl acetoacetate is dissolved in glacial acetic acid, and Mn(OAc) ₃ ·2(H ₂ O), Mn(OAc) ₂ ·4(H ₂ O) and 1,1-stilbene, wherein methyl acetoacetate: Mn(OAc) ₃ ·2(H ₂ O): Mn(OAc) ₂ ·4(H ₂ The molar ratio of O):1,1-stilbene is 1:0.02～0.05:0.02～0.05:0.5～1. React for 24 hours under oxygen conditions at room temperature. After the reaction is completed, use 6 mol/L NaOH to adjust the pH to neutral, and use saturated NaHCO ₃ solution to adjust the pH to weak alkalinity. At this time, white insoluble matter precipitated in the reaction solution, which was filtered with suction, washed with water, and washed with n-hexane to obtain compound 1.

Dissolve compound 1 in methanol, then add (1S)-(+)-camphorsulfonic acid, the molar ratio of compound 1: (1S)-(+)-camphorsulfonic acid is 1:0.2~0.6, nitrogen protection, 65°C Condensate and reflux for 24 hours. After the reaction is completed, spin the reaction liquid to dryness, add dichloromethane to dissolve the residue, wash with saturated NaHCO ₃ solution, collect the organic phase, and dry over anhydrous Na ₂ SO ₄ . Concentrate under reduced pressure to obtain a yellow oil, which is separated by silica gel column chromatography to obtain compound 2.

Dissolve compound 2 in methanol, and then add 2M NaOH aqueous solution. The molar ratio of compound 2:2M NaOH aqueous solution is 1:1~2. Under nitrogen protection, condense and reflux at 65°C overnight. After the reaction is completed, spin the reaction solution to dryness, dissolve it in deionized water, and then drop 1M HCl aqueous solution into the reaction bottle. White insoluble matter precipitates in the bottle, which is filtered, washed with water, and washed with n-hexane to obtain compound 3.

② Dissolve potassium nitrate in concentrated sulfuric acid, then add the raw material 5-methylisoxazole-3-carboxylic acid, the molar ratio of potassium nitrate to 5-methylisoxazole-3-carboxylic acid is 1:0.3-1, and react at 50°C overnight. When there is no raw material left, terminate the reaction. Transfer the reaction solution to ice water to obtain a yellow-green aqueous solution, extract with dichloromethane, collect the organic phase, dry it over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain compound 4.

Dissolve compound 4 in methylene chloride, add oxalyl chloride and DMF in sequence in an ice-water bath, the molar ratio of compound 4:oxalyl chloride:DMF is 1:2~5:0.01~0.05, and react under nitrogen protection at room temperature for 12 hours. reaction

After completion, spin the reaction liquid to dryness, add a small amount of chloroform and continue to spin the reaction liquid to dryness to obtain compound 5.

Compound 5 was dissolved in dichloromethane, and 1-Boc-piperazine and triethylamine were added in sequence, with the molar ratio of compound 5: 1-Boc-piperazine: triethylamine being 1: 1-1.2: 0.01-0.05, and the reaction was carried out under nitrogen protection at room temperature for 2 hours. After the reaction was completed, dichloromethane was added, and the mixture was washed with a saturated aqueous solution of NaHCO _3. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography to obtain a white solid 6.

Dissolve compound 6 in methylene chloride, add trifluoroacetic acid dropwise in an ice bath, stir at room temperature for 2 hours, terminate the reaction, spin the reaction solution to dryness under reduced pressure, add a small amount of methylene chloride and spin to dryness again, repeat the operation three times to remove the remaining trifluoroacetic acid. Fluoroacetic acid gave compound 7.

Compound 3 was dissolved in dichloromethane, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), diisopropylethylamine (DIPEA) and compound 7 were added in sequence, with the molar ratio of compound 3:EDCI:DIPEA:compound 7 being 1:1-1.5:1.5-2.5:1-1.2. The reaction was carried out overnight under nitrogen protection. After the reaction, the reaction solution was dissolved in dichloromethane, washed with deionized water, and the organic phase was collected and dried over _{anhydrous Na2SO4} . White solid 1b was separated by silica gel column chromatography.

③ Dissolve compound 5 in dichloromethane, add N-tert-butoxycarbonyl-1,2-ethylenediamine and triethylamine in sequence, compound 5: N-tert-butoxycarbonyl-1,2-ethylenediamine: triethylamine The molar ratio of ethylamine is 1:1~1.2:0.01~0.05, and the reaction is carried out for 2 hours under nitrogen protection at room temperature. After the reaction, add dichloromethane and wash with saturated NaHCO ₃ aqueous solution. The organic phase is collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography to obtain white solid 8.

Compound 8 was dissolved in dichloromethane, trifluoroacetic acid was added dropwise in an ice bath, and the reaction was terminated after stirring at room temperature for 2 h. The reaction solution was dried under reduced pressure, and a small amount of dichloromethane was added and dried again. The operation was repeated 3 times to remove the remaining trifluoroacetic acid to obtain compound 9.

Dissolve compound 3 in dichloromethane, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), diisopropylethylamine (DIPEA) and compound 9 in sequence. , the molar ratio of compound 3: EDCI: DIPEA: compound 9 is 1: 1 ~ 1.5: 1.5 ~ 2.5: 1 ~ 1.2. The reaction was carried out under nitrogen protection overnight. After the reaction, the reaction solution was dissolved in dichloromethane and washed with deionized water. The organic phase was collected and dried over anhydrous Na ₂ SO ₄ , and then separated by silica gel column chromatography to obtain white solid 2b.

④ Dissolve the raw material N-tert-butoxycarbonyl-1,2-ethylenediamine in tetrahydrofuran, add 4-chloro-7-nitrobenzo-2-oxa-1,3-oxadiazole and triethylamine in sequence, where , the molar ratio of N-tert-butoxycarbonyl-1,2-ethylenediamine:4-chloro-7-nitrobenzo-2-oxa-1,3-oxadiazole:triethylamine is 1:0.5～ 1: 0.01～0.05, react for 2 hours under nitrogen protection at room temperature. After the reaction was completed, the reaction liquid was spin-dried, and compound 10 was separated by silica gel column chromatography.

Dissolve compound 10 in methylene chloride, add trifluoroacetic acid dropwise in an ice bath, stir at room temperature for 2 hours, terminate the reaction, spin the reaction solution to dryness under reduced pressure, add a small amount of methylene chloride and spin to dryness again, repeat the operation three times to remove the remaining trifluoroacetic acid. Fluoroacetic acid gave compound 11.

Dissolve compound 3 in dichloromethane, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), diisopropylethylamine (DIPEA) and compound 11 in sequence. , wherein the molar ratio of compound 3: EDCI: DIPEA: compound 11 is 1:1~1.5:1.5~2.5:1~1.2. The reaction is carried out under nitrogen protection overnight. After the reaction is completed, the reaction solution is dissolved in dichloromethane and removed. Wash with ion water, collect the organic phase, dry it over anhydrous Na ₂ SO ₄ , and separate by silica gel column chromatography to obtain white solid 3b.

⑤The raw material 3-oxoheptanoic acid methyl ester is dissolved in glacial acetic acid, and Mn(OAc) ₃ ·2(H ₂ O), Mn(OAc) ₂ ·4(H ₂ O) and 1,1-stilbene are added in sequence. , where the molar ratio of methyl 3-oxoheptanoate: Mn(OAc) ₃ ·2(H ₂ O): Mn(OAc) ₂ ·4(H ₂ O): 1,1-stilbene is 1 ：0.02～0.05：0.02～0.05：0.5～1. React for 24 hours under oxygen conditions at room temperature. After the reaction is completed, use 6 mol/L NaOH to adjust the pH to neutral, and use saturated NaHCO ₃ solution to adjust the pH to weak alkalinity. At this time, white insoluble matter precipitated in the reaction solution, which was filtered with suction, washed with water, and washed with n-hexane to obtain compound 12.

Dissolve compound 12 in methanol, then add (1S)-(+)-camphorsulfonic acid, where the molar ratio of compound 12: (1S)-(+)-camphorsulfonic acid is 1:0.3~0.6, under nitrogen protection. Condensate and reflux at 65°C for 24 hours. After the reaction is completed, spin the reaction liquid to dryness, add dichloromethane to dissolve the residue, wash with saturated NaHCO ₃ solution, collect the organic phase, dry over anhydrous Na ₂ SO ₄ , and concentrate under reduced pressure to obtain a yellow oil, which is separated by silica gel column chromatography to obtain the compound. 13.

Dissolve compound 13 in methanol, and add 2M NaOH aqueous solution in sequence. The molar ratio of compound 13:2M NaOH aqueous solution is 1:1-2. Under nitrogen protection, condense and reflux at 65°C overnight. After the reaction is completed, spin the reaction solution to dryness, dissolve it in deionized water, and then drop 1 M HCl aqueous solution into the reaction bottle. White insoluble matter precipitates in the bottle, which is filtered, washed with water, and n-hexane to obtain compound 14.

⑥ Dissolve compound 14 in dichloromethane, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), diisopropylethylamine (DIPEA) and compound 11 in sequence, wherein the molar ratio of compound 14:EDCI:DIPEA:compound 11 is 1:1-1.5:1.5-2.5:1-1.2. Protect the reaction with nitrogen overnight. After the reaction, dissolve the reaction solution in dichloromethane, wash with saturated NaHCO ₃ aqueous solution, collect the organic phase, dry it over anhydrous Na ₂ SO ₄ , and separate it by silica gel column chromatography to obtain white solid 4b.

The synthesized endoperoxide compounds can be used as anti-tumor drugs or as active parts to prepare targeted anti-tumor drugs.

Beneficial effects of the present invention: The endoperoxy compounds containing GPX4 protein covalent groups according to the present invention have a certain proliferation inhibitory effect on MCF-7 cells (human breast cancer cells) in the in vitro anti-tumor activity evaluation. As potential anti-tumor compounds, develop anti-tumor drugs.

Detailed ways

The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

Example 1

Synthesis of compound 1b

(1) Dissolve 0.7 mL of commercially available methyl acetoacetate in 7 mL of glacial acetic acid, and then add 42.3 mg of Mn(OAc) ₃ ·2(H ₂ O) and 37.3 mg of Mn(OAc) ₂ ·4( H ₂ O) and 0.55 mL of commercially available 1,1-stilbene were reacted for 24 h under oxygen conditions at room temperature. After the reaction is completed, use 6 mol/L NaOH to adjust the pH to neutral, and use saturated NaHCO ₃ solution to adjust the pH to weak alkalinity. At this time, white insoluble matter precipitated in the reaction solution, which was filtered with suction, washed with water (3 × 10 mL), and washed with n-hexane (3 × 10 mL) to obtain compound 1 with a yield of 93%.

ESI-MS m/z for C ₁₉ H ₂₀ O ₅ [M+Na] ⁺ calcd 351.12, found 351.17.

(2) Weigh 366.4 mg of compound 1 and dissolve it in 10 mL of methanol, then add 39.8 mg of (1S)-(+)-camphorsulfonic acid, protect with nitrogen, and condense and reflux for 24 hours. After the reaction is completed, spin the reaction liquid to dryness, add dichloromethane to dissolve the residue, wash with saturated NaHCO ₃ solution (3 × 15 mL), collect the organic phase, and dry over anhydrous NaSO 4 . Concentrate under reduced pressure to obtain a yellow oily substance, which is separated by silica gel column chromatography. The elution condition is petroleum ether: ethyl acetate = 20:1 to obtain a pair of isomers of compound 2 with a yield of 55%.

ESI-MS m/z for C ₂₀ H ₂₂ O ₅ [M+Na] ⁺ calcd 365.14, found 365.09.

ESI-MS m/z for C ₂₀ H ₂₂ O ₅ [M+Na] ⁺ calcd 365.14, found 365.12.

(3) Dissolve 742.5 mg of compound 2 in 7 mL of methanol, then add 1.6 mL of 2M NaOH aqueous solution to the reaction bottle, protect with nitrogen, and condense and reflux at 65°C overnight. After the reaction is completed, spin the reaction solution to dryness and dissolve it with deionized water. At this time, the reaction solution is colorless and transparent. Then drop 1M HCl aqueous solution into the reaction bottle. White insoluble matter precipitates in the bottle. Filter it with water and wash with water ( 3×15mL), and washed with n-hexane (3×15mL) to obtain a pair of isomers of compound 3, with a yield of 80%.

ESI-MS m/z for C ₁₉ H ₂₀ O ₅ [MH] ^- calcd 327.12, found 327.06.

(4) 909.2 mg of commercially available potassium nitrate was dissolved in 2 mL of concentrated sulfuric acid, and then 387.3 mg of commercially available 5-methylisoxazole-3-carboxylic acid was added. After the solid was dissolved, the reaction solution was heated to 50°C, nitrogen was protected, and the reaction was allowed to proceed overnight. After no raw material was left, the reaction was terminated. The reaction solution was transferred to ice water to obtain a yellow-green aqueous solution, which was extracted with dichloromethane (3×10 mL). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow solid 4 without purification, with a yield of 70%.

(5) Dissolve 42.3 mg of compound 4 in methylene chloride, place the reaction in an ice-water bath, add 76 μL of oxalyl chloride and 30 μL of DMF in sequence, and then react under nitrogen protection at room temperature for 12 hours. After the reaction is completed, the reaction liquid is spun to dryness, a small amount of chloroform is added and the reaction liquid is further spun to dryness to obtain yellow oily substance 5. The crude product yield is 99%.

(6) 88.8 mg of compound 5 was dissolved in dichloromethane, and 85.7 mg of 1-Boc-piperazine and 50 μL of triethylamine were added in sequence, and the mixture was reacted at room temperature under nitrogen protection for 2 h. After the reaction was completed, the reaction solution was dissolved in dichloromethane, washed with saturated NaHCO ₃ aqueous solution (3×10 mL), and the organic phase was collected and dried over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a yellow oil, which was separated by silica gel column chromatography under the elution condition of petroleum ether: ethyl acetate = 2:1 to obtain a white solid 6 with a yield of 61%.

ESI-MS m/z for C ₁₄ H ₂₀ N ₄ O ₆ [M+Na] ⁺ calcd 363.1281,found 363.1282.

(7) Dissolve 76.9 mg of compound 6 in a mixed solution of trifluoroacetic acid (400 μL) and DCM (4 mL). After stirring at room temperature for 2 hours, terminate the reaction. The reaction solution is spin-dried under reduced pressure. Add a small amount of DCM and spin-dried again. Repeat the operation. The remaining trifluoroacetic acid was removed three times to obtain compound 7, with a crude product yield of 99%.

(8) Dissolve 264.9 mg of compound 3 in dichloromethane, and add 186.7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 200.2 mg of 4- Dimethylaminopyridine (DMAP), after reacting under nitrogen protection at room temperature for 1 hour, add 300 mg of compound 7 and continue the reaction overnight. After the reaction is completed, dissolve the reaction solution in dichloromethane, wash with deionized water (3 × 10 mL), and collect the organic matter The phase was dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated to obtain a yellow oil, which was separated by silica gel column chromatography. The elution conditions were petroleum ether: ethyl acetate = 2:1 to obtain compound 1b with a yield of 40%.

ESI-MS m/z for C ₂₈ H ₃₀ N ₄ O ₈ [M+Na] ⁺ calcd 573.1961, found 573.1956.

¹ H NMR (400 MHz, CDCl ₃ ) δ7.47 (d, J=6.2 Hz, 2H), 7.33 (dt, J=17.6, 8.6 Hz, 7H), 7.24 (s, 1H), 4.27 (d, J=10.7 Hz, 1H), 4.20 (d, J=10.4 Hz, 1H), 3.57-3.29 (m, 8H), 3.23 (d, J=9.3 Hz, 2H), 2.89 (s, 3H), 2.72 (d, J=5.6 Hz, 2H), 1.26 (s, 3H).

Example 2

Synthesis of compound 2b

(1) Dissolve 98.6 mg of compound 5 in dichloromethane, add 84 μL of N-tert-butoxycarbonyl-1,2-ethylenediamine and 50 μL of triethylamine in sequence, and react under nitrogen protection at room temperature for 2 hours. After the reaction is completed, dissolve the reaction solution in dichloromethane, wash with saturated NaHCO ₃ aqueous solution (3×10 mL), collect the organic phase, dry it over anhydrous sodium sulfate, filter, remove anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a yellow oil. , silica gel column chromatography, elution conditions were petroleum ether: ethyl acetate = 2:1, and compound 8 was obtained with a yield of 75%.

ESI-MS m/z for C ₁₂ H ₁₈ N ₄ O ₆ [M+Na] ⁺ calcd 337.1124, found 337.1119.

(2) Dissolve 98.1 mg of compound 8 in a mixed solution of trifluoroacetic acid (600 μL) and DCM (6 mL). After stirring at room temperature for 2 hours, terminate the reaction. The reaction solution is spin-dried under reduced pressure. Add a small amount of DCM and spin-dried again. Repeat the operation. The remaining trifluoroacetic acid was removed three times to obtain compound 9. The crude product yield was 99%.

(3) Dissolve 320.9 mg of compound 3 in dichloromethane, and add 259.8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 380 μL of diisopropyl in sequence. ethylamine (DIPEA), after reacting under nitrogen protection at room temperature for 1 hour, add compound 568.3 mg of compound 9, and continue the reaction overnight. After the reaction is completed, dissolve the reaction solution in dichloromethane, wash with deionized water (3 × 10 mL), and collect the organic The phase was dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated to obtain a yellow oil, which was separated by silica gel column chromatography. The elution conditions were petroleum ether: ethyl acetate = 1:1, and a pair of isomers of compound 2b were obtained. The rate is 67%.

ESI-MS m/z for C ₂₆ H ₂₈ N ₄ O ₈ [MH] ^- calcd 523.1829, found 523.18173.

ESI-MS m/z for C ₂₆ H ₂₈ N ₄ O ₈ [MH] ^- calcd 523.1829, found 523.18184.

¹ H NMR (400MHz, CDCl ₃ ) δ7.44 (d, J = 7.6 Hz, 3H), 7.32 (dt, J = 15.0, 8.0 Hz, 6H), 7.24 (dd, J = 6.6, 2.0 Hz, 2H) ,6.52(s,1H),3.64–3.50(m,4H),3.43(s,3H),2.97(dd,J＝18.7,6.7Hz,2H),2.83(s,3H),2.44(s,1H ),1.27(s,3H).

¹ H NMR (400MHz, CDCl ₃ ) δ7.58–7.51 (m, 2H), 7.47 (s, 1H), 7.40 (t, J = 7.6Hz, 3H), 7.29 (dd, J = 12.5, 5.0Hz, 2H),7.24–7.17(m,4H),3.62(dd,J＝9.9,4.9Hz,2H),3.54(s,5H),2.86(d,J＝9.3Hz,1H),2.82(s,3H ), 2.70 (d, J = 9.7Hz, 2H), 1.23 (s, 3H).

Example 3

Synthesis of compound 3b

(1) Dissolve 48 μL of commercially available N-tert-butoxycarbonyl-1,2-ethylenediamine in tetrahydrofuran, and add 4-chloro-7-nitrobenzo-2-oxa-1,3-oxadiazole in sequence and 50 μL triethylamine, and reacted for 2 h under nitrogen protection at room temperature. After the reaction was completed, the reaction liquid was spin-dried and separated by column chromatography. The elution conditions were petroleum ether:ethyl acetate=2:1 to obtain compound 10 with a yield of 93%.

ESI-MS m/z for C ₁₃ H ₁₇ N ₅ O ₅ [M+Na] ⁺ calcd 346.1127, found 346.11197.

(2) Dissolve 60 mg of compound 10 in a mixed solution of trifluoroacetic acid (400 μL) and DCM (4 mL). After stirring at room temperature for 2 hours, terminate the reaction. The reaction solution is spin-dried under reduced pressure. Add a small amount of DCM and spin-dried again. Repeat operation 3. The remaining trifluoroacetic acid was removed several times to obtain compound 11, with a crude product yield of 99%.

(3) 52.5 mg of compound 3 was dissolved in dichloromethane, and 36.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 55 μL of diisopropylethylamine (DIPEA) were added in sequence. After the reaction was carried out at room temperature under nitrogen protection for 1 h, 61.5 mg of compound 11 was added and the reaction was continued overnight. After the reaction was completed, the reaction solution was dissolved in DCM and washed with deionized water (3×10 mL). The organic phase was collected and dried over anhydrous Na ₂ SO ₄ , filtered, and separated by silica gel column chromatography. The elution condition was petroleum ether:ethyl acetate = 1:1 to obtain compound 3b with a yield of 41%.

ESI-MS m/z for C ₂₇ H ₂₇ N ₅ O ₇ [MH] ^- calcd 556.1808, found 556.1807.

¹ H NMR (400MHz, CDCl ₃ ) δ8.44(d,J＝8.6Hz,1H),7.72(s,1H),7.55(d,J＝7.3Hz,3H),7.42(t,J＝7.6Hz ,2H),7.32(t,J＝7.3Hz,1H),7.25–7.19(m,3H),7.14(dd,J＝7.9,1.7Hz,2H),6.17(d,J＝8.6Hz,1H) ,3.81–3.59(m,4H),3.54(s,3H),2.84(dd,J＝13.9,3.7Hz,2H),2.63(t,J＝13.9Hz,1H),1.22(s,3H).

Example 4

Synthesis of compound 4b

(1) Dissolve 357 μL of commercially available methyl 3-oxoheptanoate in 2 mL of glacial acetic acid, and then add 14.7 mg of Mn(OAc) ₃ ·2 (H ₂ O) and 13.5 mg of Mn(OAc) ₂ in sequence. ·4(H ₂ O) and 0.2 mL of commercially available 1,1-stilbene were reacted for 24 hours under oxygen conditions at room temperature. After the reaction is completed, use 6 mol/L NaOH to adjust the pH to neutral, and use saturated NaHCO ₃ solution to adjust the pH to weak alkalinity. At this time, white insoluble matter precipitated in the reaction solution, which was filtered, washed with water (3 × 10 mL), and washed with n-hexane (3 × 10 mL) to obtain compound 12, with a yield of 76%.

ESI-MS m/z for C ₂₂ H ₂₆ O ₅ [MH] ^- calcd 369.1702, found 369.17096.

(2) Weigh 310.3 mg of compound 12 and dissolve it in 10 mL of methanol, then add 28.6 mg of (1S)-(+)-camphorsulfonic acid, protect with nitrogen, and condense and reflux for 24 hours. After the reaction is completed, spin the reaction liquid to dryness, add dichloromethane to dissolve the residue, wash with saturated NaHCO ₃ solution (3 × 15 mL), collect the organic phase, and dry over anhydrous NaSO ₄ . Concentrate under reduced pressure to obtain a yellow oily substance, which is separated by silica gel column chromatography. The elution condition is petroleum ether: ethyl acetate = 30:1 to obtain compound 13 with a yield of 30%.

ESI-MS m/z for C ₂₃ H ₂₈ O ₅ [M+Na] ⁺ calcd 407.18, found 407.11.

(3) Dissolve 88 mg of compound 13 in 4 mL of methanol, then add 280 μL of 2M NaOH aqueous solution to the reaction bottle, protect with nitrogen, and condense and reflux at 65°C overnight. After the reaction is completed, spin the reaction solution to dryness and dissolve it with deionized water. At this time, the reaction solution is colorless and transparent. Then drop 1M HCl aqueous solution into the reaction bottle. White insoluble matter precipitates in the bottle. Filter it with water and wash with water ( 3×15mL), and washed with n-hexane (3×15mL) to obtain a pair of isomers of compound 14, with a yield of 45%.

ESI-MS m/z for C ₂₂ H ₂₆ O ₅ [MH] ^- calcd 369.17, found 369.09.

(4) Dissolve 64 mg of compound 14 in dichloromethane, and add 39.1 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 59 μL of diisopropyl in sequence. Ethylamine (DIPEA), after reacting under nitrogen protection at room temperature for 1 hour, add 63.5 mg of compound 11 and continue the reaction overnight. After the reaction is completed, dissolve the reaction solution in dichloromethane, wash with deionized water (3 × 10 mL), and collect the organic phase Dry over anhydrous Na ₂ SO ₄ , filter, and separate by silica gel column chromatography. The elution conditions are petroleum ether: ethyl acetate = 5:1 to obtain compound 4b with a yield of 61%.

ESI-MS m/z for C ₃₀ H ₃₃ N ₅ O ₇ [MH] ^- calcd 574.2302, found 574.2308.

¹ H NMR (600MHz, Acetone) δ8.55(d,J＝8.7Hz,1H),8.37(s,1H),7.79(s,1H),7.61–7.55(m,2H),7.41(t,J ＝7.8Hz,2H),7.33–7.19(m,6H),6.52(d,J＝8.8Hz,1H),3.83–3.63(m,4H),3.38(s,3H),2.88(dd,J＝ 12.1,2.1Hz,1H),2.81–2.70(m,2H),1.63(ddd,J＝14.3,11.6,4.8Hz,1H),1.53(ddd,J＝14.2,11.2,4.9Hz,1H),1.15 –1.06(m,1H),1.02(tt,J＝11.1,5.6Hz,2H),0.94(tdd,J＝8.2,6.0,2.7Hz,1H),0.65(t,J＝7.2Hz,3H).

Application example 1

Evaluation of the in vitro anticancer activity of the target compound of the present invention:

Compounds 1b, 2b, 3b and 4b were dissolved in dimethyl sulfoxide (cell culture grade) and stored at -20°C. When used, dilute with cell culture medium to a final concentration of dimethyl sulfoxide less than 0.1%.

MCF-7 cells (human breast cancer cells) in the logarithmic growth phase were collected and inoculated into 96-well plates (5×10 ³ cells per well), placed in an incubator (37°C, 5% CO ₂ ) and cultured for 24 hours, then the drugs were added, and blank and control groups were set up at the same time. After the cells were co-incubated with the drugs for 24 hours, 20 μL of MTT solution was added to each well, and after further culture for 4 hours, the medium was discarded, and 200 μL of dimethyl sulfoxide (chromatographic grade) was added. The absorbance at a wavelength of 570 nm was detected with an ELISA reader and the cell proliferation inhibition rate of the compound was calculated.

Table 1 Cytotoxicity of target compounds on MCF-7 cells

The above results show that target compounds 1b and 2b have a certain proliferation inhibitory effect on MCF-7 cells and can be further studied as potential anti-tumor compounds.

In summary, the present invention designed and synthesized four endoperoxide compounds containing GPX4 protein covalent groups, among which compounds 1b and 2b both had certain proliferation inhibitory effects on MCF-7 cells and could be further studied as potential anti-tumor compounds.

Claims (3)

1. An endoperoxide compound containing a GPX4 protein covalent group, characterized in that the endoperoxide compound has the activity of inhibiting tumor cell proliferation and has the following structural formulas 1b, 2b, 3b and 4b: 2. The preparation method of endoperoxide compounds as claimed in claim 1, characterized in that it includes the following steps: ①The raw material methyl acetoacetate is dissolved in glacial acetic acid, and Mn(OAc) ₃ ·2(H ₂ O), Mn(OAc) ₂ ·4(H ₂ O) and 1,1-stilbene are added in sequence, in which acetoacetic acid The molar ratio of methyl ester: Mn(OAc) ₃ ·2(H ₂ O): Mn(OAc) ₂ ·4(H ₂ O): 1,1-stilbene is 1:0.02～0.05:0.02～0.05: 0.5～1, react under room temperature oxygen conditions for 24h to obtain compound 1; Dissolve compound 1 in methanol, then add (1S)-(+)-camphorsulfonic acid, the molar ratio of compound 1: (1S)-(+)-camphorsulfonic acid is 1:0.2~0.6, nitrogen protection, 65°C Condensate and reflux for 24h to obtain compound 2; Dissolve compound 2 in methanol, and then add 2M NaOH aqueous solution. The molar ratio of compound 2:2M NaOH aqueous solution is 1:1~2. Under nitrogen protection, condense and reflux at 65°C overnight; after the reaction is completed, spin the reaction liquid to dryness. Dissolve in deionized water, and then drop 1M HCl aqueous solution into the reaction bottle to precipitate compound 3; ② Dissolve potassium nitrate in concentrated sulfuric acid, and then add the raw material 5-methylisoxazole-3-carboxylic acid. The molar ratio of potassium nitrate:5-methylisoxazole-3-carboxylic acid is 1:0.3~1. React at 50°C overnight to obtain compound 4; Dissolve compound 4 in dichloromethane, add oxalyl chloride and DMF in sequence in an ice-water bath, the molar ratio of compound 4: oxalyl chloride: DMF is 1:2~5:0.01~0.05, react under nitrogen protection at room temperature for 12 hours to obtain compound 5; Compound 5 was dissolved in dichloromethane, and 1-Boc-piperazine and triethylamine were added in sequence, with the molar ratio of compound 5: 1-Boc-piperazine: triethylamine being 1: 1-1.2: 0.01-0.05, and the mixture was reacted for 2 h under nitrogen protection at room temperature to obtain compound 6; Dissolve compound 6 in dichloromethane, add trifluoroacetic acid dropwise in an ice bath, and stir at room temperature for 2 hours to obtain compound 7; Dissolve compound 3 in dichloromethane, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI, diisopropylethylamine DIPEA and compound 7 in sequence. Compound 3: The molar ratio of EDCI:DIPEA:compound 7 is 1:1~1.5:1.5~2.5:1~1.2. The white solid 1b was obtained by reacting under nitrogen protection overnight; ③ Dissolve compound 5 in dichloromethane, add N-tert-butoxycarbonyl-1,2-ethylenediamine and triethylamine in sequence, compound 5: N-tert-butoxycarbonyl-1,2-ethylenediamine: triethylamine The molar ratio of ethylamine is 1:1~1.2:0.01~0.05. Compound 8 is obtained by reacting for 2 hours under nitrogen protection at room temperature; Dissolve compound 8 in dichloromethane, add trifluoroacetic acid dropwise in an ice bath, and stir at room temperature for 2 hours to obtain compound 9; Dissolve compound 3 in dichloromethane, add EDCI, DIPEA and compound 9 in sequence. The molar ratio of compound 3: EDCI: DIPEA: compound 9 is 1:1 ~ 1.5: 1.5 ~ 2.5: 1 ~ 1.2. The reaction was carried out under nitrogen protection overnight to obtain Compound 2b; ④ Dissolve the raw material N-tert-butoxycarbonyl-1,2-ethylenediamine in tetrahydrofuran, add 4-chloro-7-nitrobenzo-2-oxa-1,3-oxadiazole and triethylamine in sequence, where , The molar ratio of N-tert-butoxycarbonyl-1,2-ethylenediamine:4-chloro-7-nitrobenzo-2-oxa-1,3-oxadiazole:triethylamine is 1:0.5~ 1: 0.01～0.05, react at room temperature under nitrogen protection for 2 hours to obtain compound 10; Dissolve compound 10 in dichloromethane, add trifluoroacetic acid dropwise in an ice bath, and stir at room temperature for 2 hours to obtain compound 11; Dissolve compound 3 in dichloromethane, add EDCI, DIPEA and compound 11 in sequence, wherein the molar ratio of compound 3:EDCI:DIPEA:compound 11 is 1:1~1.5:1.5~2.5:1~1.2, nitrogen protection reaction White solid 3b was obtained overnight; ⑤The raw material 3-oxoheptanoic acid methyl ester is dissolved in glacial acetic acid, and Mn(OAc) ₃ ·2(H ₂ O), Mn(OAc) ₂ ·4(H ₂ O) and 1,1-stilbene are added in sequence. , where the molar ratio of methyl 3-oxoheptanoate: Mn(OAc) ₃ ·2(H ₂ O): Mn(OAc) ₂ ·4(H ₂ O): 1,1-stilbene is 1 : 0.02～0.05: 0.02～0.05: 0.5～1; Compound 12 was obtained by reacting under oxygen conditions at room temperature for 24h; Compound 12 was dissolved in methanol, and (1S)-(+)-camphorsulfonic acid was added, wherein the molar ratio of compound 12:(1S)-(+)-camphorsulfonic acid was 1:0.3-0.6, and the mixture was condensed and refluxed at 65°C for 24 hours under nitrogen protection to obtain compound 13; Dissolve compound 13 in methanol, and add 2M NaOH aqueous solution in sequence. The molar ratio of compound 13:2M NaOH aqueous solution is 1:1~2. Under nitrogen protection, condense and reflux at 65°C overnight; after the reaction is completed, spin the reaction liquid to dryness. Dissolve in deionized water, and then drop 1M HCl aqueous solution into the reaction bottle to precipitate compound 14; ⑥ Dissolve compound 14 in dichloromethane, add EDCI, DIPEA and compound 11 in sequence, wherein the molar ratio of compound 14: EDCI: DIPEA: compound 11 is 1:1~1.5:1.5~2.5:1~1.2, under nitrogen protection React overnight to obtain white solid 4b; 3. Application of the endoperoxide compound containing the covalent group of the GPX4 protein as claimed in claim 1 as an anti-human breast cancer cell drug or as an active part in the preparation of a targeted anti-human breast cancer cell drug.