GPX4 protein inhibitor as well as preparation method and application thereof

Abstract

The invention relates to a GPX4 protein inhibitor as well as a preparation method and application thereof. Specifically, the present invention relates to compounds of formula (I), pharmaceutically acceptable salts, N-oxides, hydrates, solvates, metabolites, polymorphs or prodrugs thereof, or tautomers, meso, racemes, enantiomers, diastereoisomers thereof, or mixtures thereof, the compound as shown in the formula (I) or the pharmaceutical composition thereof can selectively interfere with the activity of GPX4 so as to induce ferroptosis of tumor cells.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical application technology, and particularly to GPX4 protein inhibition, its preparation method, and its application. Specifically, this invention relates to GPX4 protein inhibitors, pharmaceutically acceptable salts, N-oxides, hydrates, solvates, metabolites, polymorphs, or prodrugs thereof, or tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, wherein the above compounds or pharmaceutical compositions thereof can effectively inhibit GPX4 protein activity, thereby inducing ferroptosis in tumor cells. Background Technology

[0002] Ferroptosis, first proposed in 2012, is an iron-dependent regulatory cell death process caused by uncontrolled lipid peroxidation and subsequent plasma membrane rupture. Ferroptosis is suppressed in various cancer types and plays a powerful tumor-suppressive role in cancer development and progression. Furthermore, a growing body of preclinical studies suggests that induced feroptosis can synergize with antitumor drugs, overcome acquired drug resistance in tumors, and activate tumor immune responses. Therefore, inducing feroptosis in tumor cells may be a promising cancer treatment strategy.

[0003] GPX4 is a selenoprotein and also a System xc - The GSH-GPX4 axis, a core protein of the major cellular system defending against ferroptosis, typically uses GSH as a cofactor. It plays a crucial role in protecting cells from lipid peroxidation and ferroptosis by reducing lipid hydroperoxides to their corresponding lipid alcohols. GPX4 is closely associated with tumorigenesis, development, and drug resistance. In the TCGA database, GPX4 expression is higher in some tumors than in normal tissues; similarly, in a general analysis of pan-cancer patients, GPX4 expression in tumor tissues was also higher than in normal tissues. Furthermore, studies have found that loss of GPX4 function leads to ferroptosis in drug-resistant tumor cells and prevents tumor recurrence in mice in vivo.

[0004] The lack of a traditional drug-forming pocket for the GPX4 protein presents challenges in designing compounds targeting it. However, small-molecule covalent inhibitors can still disrupt the enzyme's activity by irreversibly binding to selenocysteine ​​residues at the GPX4 active site. Most reported GPX4 inhibitors are alkylating agents (such as RSL3 and ML162), which covalently bind to selenocysteine ​​residues via activated alkyl chlorides. These inhibitors exhibit low selectivity and poor pharmacokinetic properties, hindering their in vivo use; currently, no GPX4 inhibitors have entered clinical trials. GPX4 has been identified as a promising therapeutic target for ferroptosis-based cancer therapy, and developing small-molecule GPX4 inhibitors to induce ferroptosis in tumor cells and effectively target cancer cells has become a hot topic in drug development.

[0005] Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a novel N-substituted tetrahydro-β-carboline, its preparation method and application, and an anti-tumor cell drug. The N-substituted tetrahydro-β-carboline provided by this invention can effectively inhibit GPX4 enzyme activity, thereby inducing ferroptosis in tumor cells.

[0007] The first aspect of this invention is to provide an inhibitor against glutathione peroxidase 4 (GPX4), characterized in that the inhibitor selectively interferes with the activity of GPX4, and the structural formula of the inhibitor is as follows:

[0008]

[0009] In formula (I), R1 is any one of the substituents shown below:

[0010]

[0011] R2 is any one of the substituents shown in b and c:

[0012]

[0013] Furthermore, the present invention claims compounds of formula (I), pharmaceutically acceptable salts thereof, N-oxides, hydrates, solvates, metabolites, polymorphs or prodrugs thereof, or tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, wherein the compounds are selected from:

[0014]

[0015] Furthermore, the pharmaceutical composition further includes other drugs that have therapeutic or preventative effects on tumors.

[0016] Furthermore, the present invention also provides the use of the GPX4 inhibitor or a pharmaceutical composition containing the GPX4 inhibitor. Specifically, as follows:

[0017] Furthermore, the present invention also provides the use of the GPX4 inhibitor or a pharmaceutical composition containing the GPX4 inhibitor in the treatment of cancer, wherein the cancer is diffuse large B-cell lymphoma, oral squamous cell carcinoma, glioma, lung cancer, colorectal cancer, bladder cancer, gastric cancer, urothelial carcinoma, prostate cancer, breast cancer, liver cancer, cervical cancer, or thyroid cancer. Further, the tumor is a tumor that highly expresses GPX4. Attached Figure Description

[0018] Figure 1 Synthetic routes of compounds

[0019] Figure 2 Effects of compounds (1S,3R)-Ab and (1S,3R)-Bb on GPX4 enzyme activity

[0020] Figure 3 Effects of compounds (1S,3R)-Ab and (1S,3R)-Bb on intracellular reactive oxygen species in HT1080 cells

[0021] Figure 4 Effects of compound (1S,3R)-Ab on malondialdehyde in HT1080 cells

[0022] Figure 5 Effect of compound (1S,3R)-Ab on the content of GPX4 enzyme in HT1080 cells

[0023] Figure 6 Effects of the compound on mouse 4T1 orthotopic tumors Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. The technical and scientific terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. The basic raw materials and reagents are obtained commercially and have a purity of 95% or higher. The present invention provides a general and specific description of the materials and experimental methods used in the experiments. Although many of the materials and methods of operation used to achieve the objectives of the present invention are well known in the art, they are still described herein as comprehensively as possible.

[0025] Example 1: Synthesis of compounds (1S,3S)-A and (1R,3S)-A

[0026]

[0027] Step 1: Synthesis of L-tryptophan methyl ester hydrochloride

[0028]

[0029] 120 mL of methanol was measured into a 250 mL flask. Under ice bath stirring, 6.4 mL of thionyl chloride (SOC12) was added dropwise using a constant pressure funnel. After drying and activation for half an hour, 5.00 g (24.5 mmol) of L-tryptophan was added to the reaction flask. The reaction was carried out at room temperature for 26 h. The reaction was stopped when the starting material spot disappeared using TLC monitoring. The reaction solution was then dried under vacuum with a water pump. 30 mL of methanol was added, and the mixture was shaken well and dried again. This process was repeated twice. Then, 30 mL of diethyl ether was added, and the ether was dried under vacuum. This process was repeated twice more to obtain 5.96 g (95.8% yield) of the title compound as a pale purple-gray solid. ESI-MS (m / e): 219.3 [M+H] + .

[0030] Synthesis of D-tryptophan methyl ester hydrochloride

[0031]

[0032] Replacing L-tryptophan with D-tryptophan, and following the same feed amounts and synthesis steps as for L-tryptophan methyl ester hydrochloride, yielded 5.77 g (92.8% yield) of the title compound as a pale purple-gray solid. ESI-MS (m / e): 219.3 [M+H] + .

[0033] Step 2: Synthesis of compounds (1S,3S)-A and (1R,3S)-A

[0034]

[0035] Weigh 5.00 g (19.6 mmol) of L-tryptophan methyl ester hydrochloride into a 250 mL flask, dissolve it in 80 mL of methanol, add 3.79 g (25.5 mmol) of cuminaldehyde, and then add 2 mL of concentrated sulfuric acid dropwise while stirring. The mixture is then heated under reflux in an oil bath for 12 h. TLC monitoring is used to monitor the reaction until the starting material spots disappear. After stopping the reaction and cooling, saturated NaHCO3 solution is slowly added dropwise with stirring in an ice bath to adjust the pH of the reaction solution to 7-8. The reaction solution is then concentrated to dryness under reduced pressure. The residue was dissolved in an appropriate amount of ethyl acetate. The ethyl acetate layer was washed three times each with saturated NaHCO3 solution and saturated NaCl solution, then dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to dryness. The crude product was purified by wet-column silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1-1 / 3) to obtain 2.60 g (yield 38.0%) of compound (1S,3S)-A and 2.37 g (yield 34.6%) of compound (1R,3S)-A, with a total yield of 72.6%, both as colorless solids. Compound (1S,3S)-A: ESI-MS (m / e): 349.4 [M+H] + ; 1 ¹H NMR (300MHz, DMSO-d⁶) δ / ppm = 10.37 (s, 1H), 7.45–7.42 (m, 1H), 7.28–7.20 (m, 5H), 7.03–6.93 (m, 2H), 5.18 (s, 1H), 3.87 (dd, J = 12.0, 6.0 Hz, 1H), 3.70 (s, 3H), 3.04 (d, J = 12.0 Hz, 1H), 2.94–2.78 (m, 2H), 2.64 (s, 1H), 1.21 (d, J = 6.0 Hz, 6H). Compound (1R, 3S)-A: ESI-MS (m / e): 349.6 [M+H] + ; 1 H NMR (300MHz, DMSO-d6) δ / ppm=10.64(s,1H),7.44(d,J=9.0Hz,1H),7.24-7.13(m,5H),7.05-6.94(m,2H),5.28(s,1H),3 .77(t,J=3.0Hz,1H),3.62(s,3H),3.14(s,1H),3.06(dd,J=15.0,3.0Hz,1H),2.91-2.81(m,2H),1.18(d,J=6.0Hz,6H).

[0036] Example 2: Synthesis of compounds (1R,3R)-A and (1S,3R)-A

[0037]

[0038] L-tryptophan methyl ester hydrochloride was replaced with D-tryptophan methyl ester hydrochloride, and the feed amount and synthesis steps were the same as those for compounds (1S,3S)-A and (1R,3S)-A. Finally, 2.40 g (yield 35.0%) of compound (1R,3R)-A and 2.18 g (yield 31.8%) of compound (1S,3R)-A were obtained, with an overall yield of 66.8%, both as colorless solids. Compound (1R,3R)-A: ESI-MS (m / e): 349.7 [M+H] + ; 1 ¹H NMR (800MHz, DMSO-d⁶) δ / ppm = 10.36 (s, 1H), 7.44 (d, J = 8.0Hz, 1H), 7.27–7.21 (m, 5H), 7.01–6.94 (m, 2H), 5.18 (s, 1H), 3.87 (dd, J = 16.0, 8.0Hz, 1H), 3.70 (s, 3H), 3.05–3.02 (m, 1H), 2.91–2.88 (m, 1H), 2.85–2.82 (m, 1H), 1.21 (d, J = 8.0Hz, 6H). Compound (1S, 3R)-A: ESI-MS (m / e): 349.6 [M+H] + ; 1 H NMR (800MHz, DMSO-d6) δ / ppm=10.63(s,1H),7.45(d,J=8.0Hz,1H),7.24-7.15(m,5H),7.00(dt,J=48.0,8.0Hz,2H) ,5.29(s,1H),3.78(t,J=8.0Hz,1H),3.63(s,3H),3.06(d,J=16.0Hz,1H),2.90-2.85(m,2H),1.18(d,J=8.0Hz,6H).

[0039] Example 3: Synthesis of compounds (1R,3R)-B and (1S,3R)-B

[0040]

[0041] Following the synthetic steps of compounds (1S,3S)-A and (1R,3S)-A, D-tryptophan methyl ester hydrochloride (2.00 g, 7.8 mmol) was weighed into a 100 mL flask, dissolved in 40 mL of methanol, followed by the addition of methyl 5-formylsalicylate (1.70 g, 9.4 mmol). Then, 1 mL of concentrated sulfuric acid was added while stirring. The mixture was then heated under reflux in an oil bath for 12 h. TLC monitoring was used to monitor the reaction until the starting material spots disappeared. The reaction was then stopped and the mixture cooled. Finally, saturated NaHCO3 solution was slowly added dropwise with stirring in an ice bath to adjust the pH of the reaction solution. The pH was adjusted to 7-8. The reaction solution was concentrated to dryness under reduced pressure. The residue was dissolved in an appropriate amount of ethyl acetate. The ethyl acetate layer was washed three times each with saturated NaHCO3 solution and saturated NaCl solution, then dried with anhydrous Na2SO4. The solution was filtered, concentrated to dryness under reduced pressure, and the crude product was purified by wet-column silica gel column chromatography (petroleum ether / ethyl acetate = 9 / 1-1 / 3) to obtain 1.02 g (yield 34.3%) of compound (1R,3R)-B and 0.93 g (yield 31.1%) of compound (1S,3R)-B, with a total yield of 65.4%, both as orange-yellow solids. Compound (1R,3R)-B: ESI-MS (m / e): 381.2 [M+H] + ; 1 ¹H NMR (300MHz, DMSO-d⁶) δ / ppm = 10.54 (s, 1H), 10.35 (s, 1H), 7.79 (d, J = 3.0Hz, 1H), 7.48 (dd, J = 9.0, 3.0Hz, 2H), 7.21 (d, J = 9.0Hz, 1H), 7.04–6.94 (m, 3H), 5.20 (s, 1H), 3.93–3.83 (m, 4H), 3.72 (s, 3H), 3.07–3.01 (m, 1H), 2.89–2.80 (m, 2H). Compound (1S, 3R)-B: ESI-MS (m / e): 381.1 [M+H] + ; 1 H NMR (300MHz, DMSO-d6) δ / ppm=10.58(s,1H),10.48(s,1H),7.66(d,J=3.0Hz,1H),7.45-7.41(m,2H),7.22(dt,J=9.0,3.0H z,1H),7.05-6.93(m,3H),5.30(s,1H),3.86-3.80(m,4H),3.62(s,3H),3.26(s,1H),3.10-3.03(m,1H),2.96-2.88(m,1H).

[0042] Example 4: Synthesis of compounds (1R,3R)-C and (1S,3R)-C

[0043]

[0044] Following the synthetic steps of compounds (1S,3S)-A and (1R,3S)-A, D-tryptophan methyl ester hydrochloride (2.00 g, 7.8 mmol) was weighed into a 100 mL flask, dissolved in 40 mL of methanol, followed by the addition of syringaldehyde (1.71 g, 9.4 mmol). Then, 1 mL of concentrated sulfuric acid was added while stirring. The mixture was then heated under reflux in an oil bath for 12 h. TLC monitoring was used to monitor the reaction until the starting material spots disappeared. After stopping the reaction and cooling, saturated NaHCO3 solution was slowly added dropwise with stirring in an ice bath to adjust the pH of the reaction solution to 7-8. The reaction solution was concentrated to dryness under reduced pressure. The residue was dissolved in an appropriate amount of ethyl acetate. The ethyl acetate layer was washed three times each with saturated NaHCO3 solution and saturated NaCl solution, then dried with anhydrous Na2SO4. After filtration and concentration to dryness under reduced pressure, the crude product was purified by dry column chromatography with silica gel (petroleum ether / ethyl acetate = 5 / 1-1 / 3) to obtain 0.97 g (yield 32.5%) of compound (1R,3R)-C as a grayish-white solid and 0.75 g (yield 25.1%) of compound (1S,3R)-C as a pale purple solid; the overall yield was 57.6%. Compound (1R,3R)-C: ESI-MS (m / e): 383.2 [M+H] + ; 1 ¹H NMR (300MHz, DMSO-d⁶) δ / ppm = 10.25 (s, 1H), 8.31 (s, 1H), 7.42 (d, J = 9.0Hz, 1H), 7.22 (d, J = 9.0Hz, 1H), 7.02–6.92 (m, 2H), 6.63 (s, 2H), 5.10 (d, J = 6.0Hz, 1H), 3.89–3.82 (m, 1H), 3.72 (s, 9H), 3.05–2.98 (m, 1H), 2.89–2.80 (m, 1H), 2.68 (t, J = 6.0Hz, 1H). Compound (1S, 3R)-C: ESI-MS (m / e): 383.2 [M+H] + ; 1 H NMR (300MHz, DMSO-d6) δ / ppm=10.50(s,1H),8.28(s,1H),7.43(d,J=9.0Hz,1H),7.24(d,J=9.0Hz,1H),7.4-6.93(m,2H) ,6.57(s,2H),5.25(s,1H),3.92(t,J=6.0Hz,1H),3.72-3.69(m,7H),3.63(s,3H),3.10-3.05(m,2H),2.96-2.89(m,1H).

[0045] Example 5: Synthesis of compounds (1R,3R)-D and (1S,3R)-D

[0046]

[0047] Following the synthetic steps of compounds (1S,3S)-A and (1R,3S)-A, 2.00 g (7.8 mmol) of D-tryptophan methyl ester hydrochloride was weighed into a 100 mL flask, dissolved in 40 mL of methanol, and then 1.43 g (9.4 mmol) of vanillin was added. 1 mL of concentrated sulfuric acid was then added while stirring. The mixture was heated under reflux in an oil bath for 12 h. TLC monitoring was used until the starting material spots disappeared. The reaction was then stopped and the mixture cooled. A saturated NaHCO3 solution was slowly added dropwise with stirring in an ice bath to adjust the pH of the reaction solution. 7-8. After concentrating the reaction solution to dryness under reduced pressure, the residue was dissolved in an appropriate amount of ethyl acetate. The ethyl acetate layer was washed three times each with saturated NaHCO3 solution and saturated NaCl solution, then dried with anhydrous Na2SO4, filtered, and concentrated to dryness under reduced pressure. The crude product was purified by dry column chromatography with silica gel (petroleum ether / ethyl acetate = 5 / 1-1 / 3) to obtain 0.98 g (yield 35.5%) of compound (1R,3R)-D and 0.76 g (yield 27.6%) of compound (1S,3R)-D, with a total yield of 63.1%, both as colorless solids. Compound (1R,3R)-D: ESI-MS (m / e): 353.2 [M+H] + ; 1 ¹H NMR (300MHz, DMSO-d⁶) δ / ppm = 10.26 (s, 1H), 8.92 (s, 1H), 7.42 (d, J = 9.0Hz, 1H), 7.22 (d, J = 9.0Hz, 1H), 7.02–6.90 (m, 3H), 6.76 (d, J = 3.0Hz, 2H), 5.11 (s, 1H), 3.88–3.83 (m, 1H), 3.72 (d, J = 3.0Hz, 6H), 3.06–2.99 (m, 1H), 2.88–2.78 (m, 1H), 2.62 (s, 1H). Compound (1S, 3R)-D: ESI-MS (m / e): 353.2 [M+H] + ; 1H NMR (300MHz, DMSO-d6) δ / ppm=10.57(s,1H),8.90(s,1H),7.43(d,J=9.0Hz,1H),7.24(d,J=9.0Hz,1H),7.05-6.94(m,3H),6.70(d,J =6.0Hz,1H),6.54(d,J=9.0Hz,1H),5.24(s,1H),3.86-3.81(m,1H),3.73(s,3H),3.63(s,3H),3.10-3.03(m,2H),2.93-2.85(m,1H).

[0048] Example 6 Synthesis of compound (1S,3S)-Ab

[0049]

[0050] Compound (1S,3S)-A (500 mg, 1.43 mmol) was weighed into a 100 mL flask and dissolved in 40 mL of dry dichloromethane. Triethylamine (240 μL, 1.72 mmol) was added, and chloroacetyl chloride (137 μL, 1.72 mmol) was slowly added dropwise with stirring in an ice bath. The reaction was carried out for 0.5 h with stirring in an ice bath. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 3:1). When the starting material spot disappeared and a new spot was formed, the reaction was stopped. The reaction solution was first quenched with water, and then washed successively with saturated sodium bicarbonate aqueous solution and saturated sodium chloride solution. The dichloromethane layer was then dried with anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated to dryness. The crude product was purified by wet column chromatography with silica gel (petroleum ether / ethyl acetate = 8 / 1-1 / 1) to obtain 559.7 mg (yield 91.8%) of the title compound as a pale yellow solid. HPLC purity: 99.59%; ESI-MS (m / e): 425.2 [M+H] + Melting point (MPa): 199.4-200.6℃; (C=0.56,CH3OH); IR (cm) -1 ):1732,1666,1625,1454,1416,1311,1287,1269,1238,1210,1180,1153,1141,1041,1006,823,739; 1H NMR (300MHz, DMSO-d6) δ / ppm=10.92(s,1H),7.56(d,J=9.0Hz,1H),7.28(d,J=9.0Hz,1H),7.17-6.98(m,6H),6.87(s,1H),5.19(d,J=9.0Hz ,1H),4.84(d,J=15.0Hz,1H),4.44(d,J=15.0Hz,1H),3.48(d,J=15.0Hz,1H),3.13-3.06(m,1H),2.89-2.75(m,4H),1.15(d,J=6.0Hz,6H). 13 C NMR(75MHz,DMSO-d6)δ / ppm=170.23,166.66,148.02,137.13,136.36,129.94,128.77,125.93,125.8 4,121.49,118.63,118.07,111.19,106.18,52.25,51.48,51.08,43.19,33.22,23.83,23.74,21.04.

[0051] Example 7 Synthesis of compound (1R,3S)-Ab

[0052]

[0053] The compound (1S,3S)-A was replaced with (1R,3S)-A, and the feed ratio and synthesis steps were the same as those for the synthesis of compound (1S,3S)-Ab. Specifically, compound (1R,3S)-A (350 mg, 1.00 mmol) was weighed into a 100 mL flask, dissolved in 40 mL of dry dichloromethane, and then triethylamine (167 μL, 1.20 mmol) was added. Chloroacetyl chloride (96 μL, 1.20 mmol) was slowly added dropwise under ice bath stirring, and the reaction was carried out for 0.5 h under ice bath stirring. After post-treatment and column chromatography purification, 426.8 mg (yield 98.3%) of the title compound was obtained as a pale yellow solid. HPLC purity: 98.70%; ESI-MS (m / e): 425.2 [M+H] + Melting point (MPa): 199.1-200.2℃; (C=0.50,CH3OH); IR (cm) -1 ):1731,1639,1451,1434,1271,1236,1224,1180,1153,825,737; 1H NMR (300MHz, DMSO-d6) δ / ppm=10.92(s,1H),7.48-6.95(m,8H),5.99(s,1H),5.38(s,1H),4.71(d,J=15 .0Hz,1H),4.39(d,J=15.0Hz,1H),3.51(s,3H),3.26-3.05(m,1H),2.82(s,1H),1.16(d,J=6.0Hz,6H). 13 C NMR (75MHz, DMSO-d6) δ / ppm=171.62,167.31,136.04,126.49,125.53,120.93,118.54,117.64,111.04,56.39,52.43,43.06,32.75,23.49.

[0054] Example 8: Synthesis of compound (1R,3R)-Ab

[0055]

[0056] The compound (1S,3S)-A was replaced with (1R,3R)-A, and the feed ratio and synthesis steps were the same as those for the synthesis of compound (1S,3S)-Ab. Specifically, 500 mg (1.43 mmol) of compound (1R,3R)-A was weighed into a 100 mL flask, dissolved in 40 mL of dry dichloromethane, and then triethylamine (240 μL, 1.72 mmol) was added. Chloroacetyl chloride (137 μL, 1.72 mmol) was slowly added dropwise under ice bath stirring, and the reaction was carried out for 0.5 h under ice bath stirring. After post-treatment and column chromatography purification, 570.1 mg (yield 93.5%) of the title compound was obtained as a grayish-white solid. HPLC purity: 99.75%; ESI-MS (m / e): 447.3 [M+Na] + Melting point (MPa): 196.8-197.2℃; (C=0.55,CH3OH); IR (cm) -1 ):1732,1656,1416,1310,1287,1269,1258,1210,1179,1153,1140,822,747; 1H NMR (300MHz, DMSO-d6) δ / ppm=10.91(s,1H),7.55(d,J=6.0Hz,1H),7.28(d,J=9.0Hz,1H),7.16-6.97(m,6H),6.86(s,1H),5.19(d,J=6.0Hz,1H) ,4.83(d,J=15.0Hz,1H),4.43(d,J=15.0Hz,1H),3.47(d,J=15.0Hz,1H),3.09(dd,J=15.0,6.0Hz,1H),2.87-2.78(m,4H),1.15(d,J=9.0Hz,6H). 13 CNMR(75MHz,DMSO-d6)δ / ppm=170.50,166.92,148.28,137.41,136.63,130.21,129.04,126.20,126. 10,121.74,118.89,118.33,111.46,106.45,52.52,51.73,51.36,43.44,33.47,24.08,23.99,21.30.

[0057] Example 9: Synthesis of compound (1S,3R)-Ab

[0058]

[0059] The compound (1S,3S)-A was replaced with (1S,3R)-A, and the feed ratio and synthesis steps were the same as those for the synthesis of compound (1S,3S)-Ab. Specifically, 350 mg (1.00 mmol) of compound (1S,3R)-A was weighed into a 100 mL flask, dissolved in 40 mL of dry dichloromethane, and then triethylamine (167 μL, 1.20 mmol) was added. Chloroacetyl chloride (96 μL, 1.20 mmol) was slowly added dropwise under ice bath stirring, and the reaction was carried out for 0.5 h under ice bath stirring. After post-treatment and column chromatography purification, 394.4 mg (yield 92.4%) of the title compound was obtained as a pale yellow solid. HPLC purity: 98.05%; ESI-MS (m / e): 447.2 [M+Na] + Melting point (MPa): 200.9-201.6℃; (C=0.53,CH3OH); IR (cm) -1 ):1729,1638,1451,1435,1349,1271,1236,1225,1154,1047,825,736; 1H NMR(300MHz,DMSO-d6)δ / ppm=10.98(s,1H),7.48-6.95(m,8H),6.28-6.00(m,1H),5.38-4.88(m,1H),4 .70(d,J=15.0Hz,1H),4.41-4.17(m,1H),3.52(s,3H),3.21(s,1H),2.83(s,1H),1.16(d,J=6.0Hz,6H). 13 C NMR (75MHz, DMSO-d6) δ / ppm=172.01,167.86,136.61,135.08,127.04,126.08,121.48,119. 09,118.17,111.59,103.49,56.93,56.20,54.36,52.93,52.26,43.63,43.05,33.30,24.04.

[0060] Example 10 Synthesis of compound (1S,3S)-Ac

[0061]

[0062] Chloroacetyl chloride was replaced with acryloyl chloride, and the feed ratio and synthesis steps were the same as those for compound (1S,3S)-Ab. Specifically, compound (1S,3S)-A (350 mg, 1.00 mmol) was weighed into a 100 mL flask, dissolved in 40 mL of dry dichloromethane, and then triethylamine (167 μL, 1.20 mmol) was added. Acryloyl chloride (99 μL, 1.20 mmol) was slowly added dropwise under ice bath stirring, and the reaction was carried out for 0.5 h under ice bath stirring. After post-treatment and column chromatography purification, 369.5 mg (yield 91.4%) of the title compound was obtained as a pale yellow solid. HPLC purity: 99.51%; ESI-MS (m / e): 403.6 [M+H] + Melting point (MPa): 192.1-193.2℃; (C=0.50,CH3OH); IR (cm) -1 ):1741,1635,1609,1428,1311,1271,1238,1209,1178,1154,998,902,746; 1H NMR (300MHz, DMSO-d6) δ / ppm=10.94(s,1H),7.53(d,J=9.0Hz,1H),7.28(d,J=6.0Hz,1H),7.20-6.93(m,8H),6.26(d,J=18.0Hz,1 H),5.80(d,J=12.0Hz,1H),5.44(d,J=6.0Hz,1H),3.46(d,J=18.0Hz,1H),3.01-2.93(m,2H),2.81(s,3H),1.15(d,J=9.0Hz,6H). 13 C NMR(75MHz,DMSO-d6)δ / ppm=170.63,166.54,147.87,137.52,136.36,130.31,128.80,128.63,127.62,12 6.03,125.81,121.42,118.61,118.06,111.19,106.25,52.05,51.37,50.37,33.22,23.86,23.76,21.50.

[0063] Example 11 Synthesis of compound (1R,3S)-Ac

[0064]

[0065] Chloroacetyl chloride was replaced with acryloyl chloride, and the feed ratio and synthesis steps were the same as those for compound (1S,3S)-Ab. Specifically, compound (1R,3S)-A (350 mg, 1.00 mmol) was weighed into a 100 mL flask, dissolved in 40 mL of dry dichloromethane, and triethylamine (167 μL, 1.20 mmol) was added. Acryloyl chloride (99 μL, 1.20 mmol) was slowly added dropwise under ice bath stirring, and the reaction was carried out for 0.5 h under ice bath stirring. After post-treatment and column chromatography purification, 376.8 mg (yield 93.2%) of the title compound was obtained as a pale yellow solid. HPLC purity: 98.24%; ESI-MS (m / e): 403.6 [M+H] + Melting point (MPa): 203.2-203.7℃; (C=0.54,CH3OH); IR (cm) -1 ):1732,1640,1605,1449,1434,1344,1271,1218,1175,1153,1095,1054,973,825,737; 1H NMR (300MHz, DMSO-d6) δ / ppm=10.98(s,1H),7.47-6.74(m,9H),6.31(s,1H),6.14(d,J=18.0Hz,1H),5.67(d,J =9.0Hz,1H),4.88(d,J=9.0Hz,1H),3.53(s,3H),3.29-3.12(m,1H),2.88-2.80(m,1H),1.16(d,J=6.0Hz,6H). 13 CNMR(75MHz,DMSO-d6)δ / ppm=170.95,167.15,147.88,139.19,136.29,133.44,129.09,128.60,126.75,126.6 3,126.16,125.83,121.35,118.85,117.92,111.36,105.42,56.43,53.58,51.93,33.04,23.84,23.80,22.24.

[0066] Example 12 Synthesis of compound (1R,3R)-Ac

[0067]

[0068] Chloroacetyl chloride was replaced with acryloyl chloride, and the feed ratio and synthesis steps were the same as those for compound (1S,3S)-Ab. Specifically, compound (1R,3R)-A (205 mg, 0.59 mmol) was weighed into a 50 mL flask, dissolved in 25 mL of dry dichloromethane, and triethylamine (98 μL, 0.70 mmol) was added. Acryloyl chloride (56 μL, 0.70 mmol) was slowly added dropwise under ice bath stirring, and the reaction was carried out for 0.5 h under ice bath stirring. After post-treatment and column chromatography purification, 214.0 mg (yield 90.4%) of the title compound was obtained as a pale yellow solid. HPLC purity: 99.06%; ESI-MS (m / e): 403.6 [M+H] + Melting point (MPa): 194.8-195.3℃; (C=0.54,CH3OH); IR (cm) -1 ):1741,1634,1610,1428,1311,1271,1209,1177,1154,1144,999,953,901,855,763,721; 1H NMR (300MHz, DMSO-d6) δ / ppm=10.93(s,1H),7.54(d,J=9.0Hz,1H),7.30-6.93(m,9H),6.26(d,J=15.0Hz,1H),5.80(d,J =9.0Hz,1H),5.44(d,J=6.0Hz,1H),3.47(d,J=15.0Hz,1H),3.03-2.95(m,2H),2.85-2.81(m,4H),1.15(d,J=6.0Hz,6H). 13 C NMR(75MHz,DMSO-d6)δ / ppm=169.78,166.68,150.89,140.85,137.06,129.04,126.05,121.6 7,118.85,118.30,111.44,106.49,52.31,51.61,50.63,33.48,26.62,24.11,24.02,21.76.

[0069] Example 13 Synthesis of compound (1S,3R)-Ac

[0070]

[0071] Chloroacetyl chloride was replaced with acryloyl chloride, and the feed ratio and synthesis steps were the same as those for compound (1S,3S)-Ab. Specifically, compound (1S,3R)-A (150 mg, 0.43 mmol) was weighed into a 50 mL flask, dissolved in 20 mL of dry dichloromethane, and triethylamine (71 μL, 0.51 mmol) was added. Acryloyl chloride (41 μL, 0.51 mmol) was slowly added dropwise under ice bath stirring, and the reaction was carried out for 0.5 h under ice bath stirring. After post-treatment and column chromatography purification, 163.5 mg (yield 94.4%) of the title compound was obtained as a pale yellow solid. HPLC purity: 99.44%; ESI-MS (m / e): 403.7 [M+H] + Melting point (MPa): 211.6-212.4℃; (C=0.56,CH3OH); IR (cm) -1 ):1742,1639,1605,1450,1434,1345,1269,1234,1220,1157,966,826,792,737; 1H NMR (300MHz, DMSO-d6) δ / ppm=10.98(s,1H),7.48-6.74(m,9H),6.32(s,1H),6.15(d,J=18.0Hz,1H),5.68(d,J=9 .0Hz,1H),4.90(s,1H),3.51-3.49(m,3H),3.16(d,J=18.0Hz,1H),2.83(d,J=9.0Hz,1H),1.15(d,J=9.0Hz,6H). 13 CNMR(75MHz,DMSO-d6)δ / ppm=170.93,167.14,147.85,140.13,136.29,134.25,129.25,128.32,126.74,126.6 0,126.14,125.83,122.07,118.84,117.90,111.35,105.44,56.43,53.61,51.92,33.03,23.82,23.78,22.39.

[0072] Example 14 Synthesis of compound (1R,3R)-Bb

[0073]

[0074] Compound (1S,3S)-A was replaced with (1R,3R)-B, and the feed ratio and synthesis steps were the same as those for compound (1S,3S)-Ab. Specifically, compound (1R,3R)-B (380 mg, 1.00 mmol) was weighed into a 100 mL flask, dissolved in 40 mL of dry dichloromethane, and then triethylamine (167 μL, 1.20 mmol) was added. Chloroacetyl chloride (96 μL, 1.20 mmol) was slowly added dropwise under ice bath stirring, and the reaction was carried out for 0.5 h under ice bath stirring. After post-treatment and column chromatography purification, 420.8 mg (yield 92.1%) of the title compound was obtained as a grayish-white solid. HPLC purity: 99.68%; ESI-MS (m / e): 474.3 [M+NH4] + Melting point (MPa): 168.3-169.5℃; (C=0.54,CH3OH); IR (cm) -1 ):3395,1741,1676,1652,1423,1307,1263,1236,1201,1177,1156,1090,794,750,673; 1H NMR(300MHz,DMSO-d6)δ / ppm=10.82(s,1H),10.52(s,1H),7.61-7.55(m,2H), 7.29(d,J=6.0Hz,1H),7.19(dd,J=9.0,3.0Hz,1H),7.14-7.01(m,2H),6.93(d ,J=9.0Hz,1H),6.74(s,1H),5.23(d,J=6.0Hz,1H),4.85(d,J=15.0Hz,1H),4. 44(d,J=15.0Hz,1H),3.84(s,3H),3.50(d,J=18.0Hz,1H),3.14-3.05(m,4H). 13 C NMR(75MHz,DMSO-d6)δ / ppm=170.38,168.99,166.86,159.51,136.48,136.19,130.65,130.48,129.73,12 5.91,121.63,118.73,118.19,116.98,112.46,111.31,106.43,54.89,52.51,51.81,51.20,43.20,21.13.

[0075] Example 15 Synthesis of compound (1S,3R)-Bb

[0076]

[0077] Compound (1S,3S)-A was replaced with (1S,3R)-B, with the same feed ratio and synthetic steps as compound (1S,3S)-Ab. Specifically, compound (1S,3R)-B (380 mg, 1.00 mmol) was weighed into a 100 mL flask, dissolved in 40 mL of dry dichloromethane, and then triethylamine (167 μL, 1.20 mmol) was added. Chloroacetyl chloride (96 μL, 1.20 mmol) was slowly added dropwise under ice bath stirring, and the reaction was carried out for 0.5 h under ice bath stirring. After post-treatment and column chromatography purification, 409.3 mg (yield 89.6%) of the title compound was obtained as a pale yellow solid. HPLC purity: 99.21%; ESI-MS (m / e): 474.3 [M+NH4] + Melting point (MPa): 130.1-130.9℃; (C=0.51,CH3OH); IR (cm) -1 ):3289,1732,1667,1488,1439,1302,1270,1209,1155,1087,794,742,691; 1H NMR (300MHz, DMSO-d6) δ / ppm=10.88(s,1H),10.41(s,1H),7.85(d,J=3.0Hz,1H),7.69-7.48(m,2H),7.35-7.15(m,1H),7.04-6 .84(m,3H),5.94(s,1H),5.36(s,1H),4.70(d,J=15.0Hz,1H),4.41-4.20(m,1H),3.88(s,3H),3.61-3.52(m,4H),3.21(s,1H). 13 C NMR (75MHz, DMSO-d6) δ / ppm=172.16,169.56,168.37,159.38,136.89,134.94,134.16,128.80,126.24 ,121.80,119.34,118.54,117.71,112.84,111.83,103.89,60.23,57.07,53.25,52.95,43.83,21.24.

[0078] Example 16 Synthesis of compound (1R,3R)-Cb

[0079]

[0080] Compound (1R,3R)-C (589 mg, 1.54 mmol) was weighed into a 100 mL flask and dissolved in 40 mL of dry dichloromethane. Sodium bicarbonate powder (143 mg, 1.70 mmol) was added, and chloroacetyl chloride (133 μL, 1.70 mmol) was slowly added dropwise with stirring in an ice bath. The reaction was carried out for 0.5 h with stirring in an ice bath. The reaction was monitored by TLC (petroleum ether:ethyl acetate = 1:1). The reaction was stopped when the starting material spot disappeared and a new spot formed. The reaction solution was first quenched with water, then washed successively with saturated sodium bicarbonate aqueous solution and saturated sodium chloride solution. The dichloromethane layer was then collected, dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated to dryness. The crude product was purified by dry column chromatography with silica gel (petroleum ether / ethyl acetate = 5 / 1-1 / 2) to obtain 655.8 mg (yield 92.9%) of the title compound as an orange-red solid. HPLC purity: 96.52%; ESI-MS (m / e): 497.1 [M+K] + Melting point (MPa): 112.0-113.3℃; (C=0.54,CH3OH); IR (cm) -1 ):3356,1741,1642,1607,1513,1455,1427,1370,1306,1203,1107,901,840,739;1 H NMR (300MHz, DMSO-d6) δ / ppm=10.87(s,1H),8.44(s,1H),7.55(d,J=9.0Hz,1H),7.29(d,J=9.0Hz,1H),7.12-7.00(m,2H),6.76(s,1H), 6.38(s,2H),5.20(d,J=6.0Hz,1H),4.83(d,J=12.0Hz,1H),4.44(d,J=12.0Hz,1H),3.74(s,1H),3.59-3.46(m,6H),3.13-3.02(m,4H). 13 C NMR(75MHz,DMSO-d6)δ / ppm=170.66,166.71,147.47,136.45,135.75,130.25,129.56,125.86 ,121.50,118.62,118.15,111.25,107.15,106.13,59.77,56.05,52.29,51.69,43.21,21.02.

[0081] Example 17 Synthesis of compound (1S,3R)-Cb

[0082]

[0083] The compound (1R,3R)-C was replaced with (1S,3R)-C, and the feed ratio and synthesis steps were the same as those for the synthesis of compound (1R,3R)-Cb. Specifically, compound (1S,3R)-C (382 mg, 1.00 mmol) was weighed into a 100 mL flask, dissolved in 40 mL of dry dichloromethane, and sodium bicarbonate powder (93 mg, 1.10 mmol) was added. Chloroacetyl chloride (86 μL, 1.10 mmol) was slowly added dropwise under ice bath stirring, and the reaction was carried out for 0.5 h under ice bath stirring. After post-treatment and column chromatography purification, 411.7 mg (yield 89.9%) of the title compound was obtained as an orange solid. HPLC purity: 95.87%; ESI-MS (m / e): 497.1 [M+K] + Melting point (MPa): 138.6-139.7℃; (C=0.47,CH3OH); IR (cm) -1 ):3301,1732,1655,1454,1424,1209,1153,1110,1041,744; 1H NMR (300MHz, DMSO-d6) δ / ppm=10.98(s,1H),8.27(s,1H),7.46(d,J=9.0Hz,1H),7.29(s,1H),7.00(dt,J=18.0,9.0Hz,2H),6.73(d,J=18. 0Hz,2H),6.16-5.93(m,1H),5.37-5.01(m,1H),4.69-4.64(m,1H),4.31-4.13(m,1H),3.75(s,6H),3.55-3.51(m,4H),3.28-3.23(m,1H). 13 C NMR(75MHz,DMSO-d6)δ / ppm=170.32,167.81,148.19,136.26,133.24,131.79,130.42,125.77,12 1.21,118.77,117.91,111.29,104.17,103.04,59.72,56.81,56.11,53.95,51.28,43.15,20.72.

[0084] Example 18 Synthesis of compound (1R,3R)-Cbb

[0085]

[0086] Weigh 765 mg (2.00 mmol) of the unpurified diastereomer (1S / R, 3R)-C into a 100 mL flask, dissolve it in 40 mL of dry dichloromethane, add triethylamine (556 μL, 4.00 mmol), and slowly add chloroacetyl chloride (235 μL, 3.00 mmol) dropwise while stirring in an ice bath. React for 0.5 h while stirring in an ice bath. Monitor the reaction by TLC (petroleum ether:ethyl acetate = 1:1). When the starting material spot disappears and a new spot forms, stop the reaction. First, quench the reaction with water, then wash the reaction solution successively with saturated sodium bicarbonate aqueous solution and saturated sodium chloride solution. Then, take the dichloromethane layer, dry it with anhydrous sodium sulfate, filter under reduced pressure, concentrate the filtrate to dryness, and purify the crude product by silica gel column chromatography (petroleum ether / ethyl acetate = 8 / 1-1 / 2) to obtain 160.7 mg (yield 30.1%) of the title compound as a colorless solid. HPLC purity: 100.0%; ESI-MS (m / e): 552.3 [M+NH4] + Melting point (MPa): 204.5-205.8℃; (C=0.51,CH3OH); IR (cm) -1):1784,1745,1650,1604,1506,1467,1455,1421,1319,1251,1237,1213,1127,1006,901,764; 1 H NMR (300MHz, DMSO-d6) δ / ppm=10.98(s,1H),7.58(d,J=9.0Hz,1H),7.31(d,J =6.0Hz,1H),7.14-7.01(m,3H),6.84(s,1H),6.50(s,2H),5.25(d,J=9.0Hz, 1H),4.86(d,J=15.0Hz,1H),4.73(s,2H),4.48(d,J=15.0Hz,1H),3.76(s,1H ),3.59(s,6H),3.53-3.48(m,2H),3.12(dd,J=15.0,6.0Hz,1H),3.04(s,3H). 13 C NMR(75MHz,DMSO-d6)δ / ppm=170.66,167.02,165.57,151.11,138.62,136.51,129.37,127.19,125.79,1 21.74,118.75,118.31,111.35,106.50,105.69,55.91,54.94,52.45,51.73,51.68,43.17,40.65,20.99.

[0087] Example 19 Synthesis of compound (1R,3R)-Db

[0088]

[0089] Compound (1R,3R)-D (350 mg, 1.00 mmol) was weighed into a 100 mL flask and dissolved in 40 mL of dry dichloromethane. Sodium bicarbonate powder (93 mg, 1.10 mmol) was added, and chloroacetyl chloride (88 μL, 1.10 mmol) was slowly added dropwise with stirring in an ice bath. The reaction was carried out for 0.5 h with stirring in an ice bath. The reaction was monitored by TLC (petroleum ether:ethyl acetate = 1:1). The reaction was stopped when the starting material spot disappeared and a new spot formed. The reaction solution was first quenched with water, then washed successively with saturated sodium bicarbonate aqueous solution and saturated sodium chloride solution. The dichloromethane layer was then collected, dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated to dryness. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1-1 / 2) to obtain 395.1 mg (yield 92.3%) of the title compound as an orange-yellow solid. HPLC purity: 97.62%; ESI-MS (m / e): 467.1 [M+K] +Melting point (MPa): 113.2-113.7℃; (C=0.46,CH3OH); IR (cm) -1 ):3304,1731,1643,1510,1429,1309,1270,1204,1163,1031,792,740,675; 1 H NMR (300MHz, DMSO-d6) δ / ppm=10.87(s,1H),9.01(s,1H),7.54(d,J=9.0Hz,1H),7. 28(d,J=9.0Hz,1H),7.12-7.00(m,2H),6.77-6.75(m,2H),6.65(d,J=9.0Hz,1H),6. 39(d,J=9.0Hz,1H),5.19(d,J=6.0Hz,1H),4.83(d,J=12.0Hz,1H),4.44(d,J=12.0 Hz,1H),3.62(s,3H),3.50-3.44(m,1H),3.08(dd,J=15.0,6.0Hz,1H),2.98(s,3H). 13 C NMR(75MHz,DMSO-d6)δ / ppm=170.49,166.66,147.02,146.42,136.37,130.51,130.37,125.90,121.73 ,121.44,118.59,118.07,114.72,113.61,111.20,106.04,59.76,55.67,52.23,51.67,43.20,21.03.

[0090] Example 20 Synthesis of compound (1S,3R)-Db

[0091]

[0092] The compound (1R,3R)-D was replaced with (1S,3R)-D, and the feed ratio and synthesis steps were the same as those for the synthesis of compound (1R,3R)-Db. Specifically, 350 mg (1.00 mmol) of compound (1R,3R)-D was weighed into a 100 mL flask, dissolved in 40 mL of dry dichloromethane, and then 93 mg (1.10 mmol) of sodium bicarbonate powder was added. Chloroacetyl chloride (88 μL, 1.10 mmol) was slowly added dropwise under ice bath stirring, and the reaction was carried out for 0.5 h under ice bath stirring. After post-treatment and column chromatography purification, 379.3 mg (yield 88.6%) of the title compound was obtained as a powdery white solid. HPLC purity: 96.40%; ESI-MS (m / e): 467.1 [M+K] +Melting point (MPa): 132.5-133.4℃; (C=0.48,CH3OH); IR (cm) -1 ):3337,1731,1645,1512,1452,1429,1372,1270,1236,1203,1123,1030,1009,792,739,676; 1 H NMR (300MHz, DMSO-d6) δ / ppm=10.89(s,1H),8.77(s,1H),7.46(d,J=6.0Hz,1H),7.28-6.63(m,6H),6.17(s ,1H),4.88(s,1H),4.70(d,J=15.0Hz,1H),4.34-4.16(m,1H),3.77(s,3H),3.54(s,3H),3.27-3.18(m,2H). 13 C NMR (75MHz, DMSO-d6) δ / ppm=170.68,167.68,148.83,146.12,136.29,133.97,130.87,125.82,121.23 ,119.18,118.78,117.92,115.52,111.31,111.05,104.58,59.77,56.79,55.71,54.00,43.20,20.77.

[0093] Example 21 Synthesis of compound (1R,3R)-Dbb

[0094]

[0095] Compound (1R,3R)-D (350 mg, 1.00 mmol) was weighed into a 50 mL flask and dissolved in 25 mL of dry dichloromethane. Triethylamine (278 μL, 2.00 mmol) was added, and chloroacetyl chloride (118 μL, 1.50 mmol) was slowly added dropwise with stirring in an ice bath. The reaction was carried out for 0.5 h with stirring in an ice bath. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 1:1). The reaction was stopped when the starting material spot disappeared and a new spot was formed. The reaction solution was first quenched with water, and then washed successively with saturated sodium bicarbonate aqueous solution and saturated sodium chloride solution. The dichloromethane layer was then dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated to dryness. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1-1 / 2) to give 457.9 mg (yield 91.2%) of the title compound as a colorless solid. HPLC purity: 93.02%; ESI-MS (m / e): 522.0 [M+NH4] +Melting point (MPa): 181.3-182.0℃; (C=0.49,CH3OH); IR (cm) -1 ):3282,1788,1747,1645,1504,1452,1424,1415,1310,1266,1250,1237,1211,1126,1034,921,746,666; 1 H NMR (300MHz, DMSO-d6) δ / ppm=10.98(s,1H),7.57(d,J=6.0Hz,1H),7.30(d,J =9.0Hz,1H),7.14-7.01(m,3H),6.97(s,1H),6.87(s,1H),6.57(d,J=9.0Hz, 1H),5.24(d,J=6.0Hz,1H),4.86(d,J=12.0Hz,1H),4.71(s,2H),4.48(d,J=1 2.0Hz,1H),3.63(s,3H),3.52-3.46(m,1H),3.16-3.08(m,1H),2.97(s,3H). 13 C NMR(75MHz,DMSO-d6)δ / ppm=170.38,166.99,165.86,150.16,139.09,138.46,136.42,129.43,125.84,122.0 6,121.68,121.10,118.73,118.22,113.40,111.30,106.44,55.72,52.39,51.70,51.18,43.15,40.91,21.01.

[0096] Example 22 Synthesis of compound (1S,3S)-Eb

[0097]

[0098] Following the synthetic method for compound (1S,3S)-Ab, replacing cinnamaldehyde with cuminaldehyde yielded the title compound. ESI-MS (m / e) 431.0 [M+Na] + ; 1H-NMR(300MHz DMSO-d6): δ / ppm=10.90(d,J=12.76Hz,1H),7.54-7.27(m,9H),7.12-6.99(m,2H),5.88(d,J=9Hz,1H),5 .27(d,J=6Hz,1H),4.91(dd,J=15.0,42Hz,1H),4.60-4.48(m,1H),3.48(s,2H),3.42(s,3H),3.10(m,1H)

[0099] Example 23 Synthesis of compound (1R,3S)-Eb

[0100]

[0101] Following the synthetic method of compound (1R,3S)-Ab, replacing cinnamaldehyde with cuminaldehyde yielded the title compound. ESI-MS (m / e) 431.2 [M+Na] + ; 1 H-NMR(300MHz DMSO-d6): δ / ppm=11.07(s,1H),7.50-6.97(m,9H),6.66(d,J=27Hz,2H),5.85(s, 1H),4.71(d,J=12Hz,2H),4.53(d,J=12Hz,1H),3.59(s,3H),3.15(d,J=21Hz,2H)

[0102] Example 24 Synthesis of compound (1R,3R)-Eb

[0103]

[0104] Following the synthetic method of compound (1R,3R)-Ab, replacing cinnamaldehyde with cuminaldehyde yielded the title compound. ESI-MS (m / e) 431.2 [M+Na] + ; 1 H-NMR(300MHz DMSO-d6): δ / ppm=10.92(d,J=9.3Hz,1H),7.51-7.34(m,9H),7.10-7.01(m,2H),6.65-6.43(m,1H),6.60(d,J=13 .7Hz,1H),5.00-4.80(m,1H),4.60-4.48(t,J=12Hz,1H),3.48(s,2H),3.33(s,3H),3.01(dd,J=12.0,14.0Hz,2H)

[0105] Example 25 Synthesis of compound (1S,3R)-Eb

[0106]

[0107] Following the synthetic method of compound (1S,3R)-Ab, replacing cinnamaldehyde with cuminaldehyde yielded the title compound. ESI-MS (m / e) 431.2 [M+Na] + ; 1 H-NMR(300MHz DMSO-d6): δ / ppm=11.06(s,1H),7.50-7.31(m,9H),7.07-6.99(m,2H),6.70(d,J=14.2Hz,2H),5.85( d,J=13.5Hz,1H),4.70(d,J=10.6Hz,2H),4.49(d,J=9.9Hz,1H),3.59(s,3H),3.09(d,J=12.4Hz,2H)

[0108] Example 26 Synthesis of compound (1S,3R)-Fb-1

[0109]

[0110] Following the synthetic method for compound (1S,3R)-Ab, replacing cuminaldehyde with N-cyclopropyl-4-formylbenzamide yielded the title compound. ESI-MS (m / e) 461.1 [M+H] + ; 1 H-NMR(300MHz DMSO-d6): δ / ppm=10.91(s,1H),8.27(s,1H),7.65(d,J=7.29Hz,2H),7.47(d,J=7.44Hz,2H),7.22(d,J=7.44Hz,1H),6.01 (s,1H),5.39(s,1H),4.74(s,1H),4.70(s,1H),3.51(s,3H),3.38-3.31(m,2H),2.79(m,J=3.78Hz,2H),0.66-0.51(m,4H)

[0111] Example 27 Synthesis of compound (1S,3R)-Fb-2

[0112]

[0113] The title compound was synthesized using the same method as compound (1S,3R)-Fb-2. ESI-MS (m / e) 466.2 [M+H] + ; 1H-NMR(300MHz DMSO-d6): δ / ppm=11.03(s,1H),8.10(s,1H),7.80(d,J=6.99Hz,2H),7.61(d,J=7.86Hz,2H),7.40(d,J=6.99Hz,2H),7.25(d,J=7.86Hz,1 H),6.91-7.02(m,2H),6.24(s,1H),5.70(s,1H),5.23(s,1H),5.12(s,1H),3.77(s,3H),3.60-3.45(m,2H),2.42(m,1H),0.46-0.28(m,4H)

[0114] Evaluation of the antitumor cell proliferation activity of compound in Test Example 1

[0115] PBS and culture medium were purchased from Jiangsu Kaiji Biotechnology Co., Ltd., fetal bovine serum (FBS) was purchased from CORNING, dimethyl sulfoxide (DMSO) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and tetrathiazolyl blue (MTT) was purchased from Solarbio. The solutions were dissolved in PBS to prepare a 5 mg / mL solution, filtered for sterilization, and stored in the dark.

[0116] Test cell lines: 4T1 (mouse breast cancer cells), MCF-7 (human breast cancer cells), A549 (human non-small cell lung cancer cells), HT1080 (human fibrosarcoma cells), LLC (mouse Lewis lung cancer cells);

[0117] 4T1 and A549 cells were cultured in RPMI-1640 medium, while MCF-7 and LLC cells were cultured in DMEM medium containing 10% fetal bovine serum. HT1080 cells were cultured in HT1080 complete medium from Wuhan Pronosai Company.

[0118] Cells in the logarithmic growth phase were washed three times with PBS, then digested with trypsin until most cells detached from the flask wall. The digestion was stopped by adding the appropriate culture medium, and the cells were pipetted until completely detached. The cells were transferred to 15 mL centrifuge tubes and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in culture medium and counted. The cell density was 3–4 × 10⁶ cells / mL. 4Cells were seeded at a concentration of 100 μL / mL into 96-well plates (Corning 3599), with 100 μL of PBS added to each well (100 μL of PBS was added to the periphery of each well for sealing). Different test compounds were added for incubation. Cells were incubated at 37°C and 5% CO2 for 6 hours, and cell adhesion was observed. When the adhesion rate reached 50% or higher, 25 μL of sample solution was added to each well and gently shaken to mix. After 48 hours of incubation, 25 μL of 5 mg / mL MTT solution was added to each well, and incubation was continued for 4 hours. The supernatant was removed, and 100 μL of DMSO was added to each well. The mixture was shaken for 15 minutes to fully dissolve the precipitate. The absorbance of each well was measured at 570 nm and 490 nm using a microplate reader within 5 minutes. Triple replicates were used. Inhibition rate was plotted against the concentration of the test compound, and IC50 was calculated. 50 value.

[0119] The experimental results are shown in Table 1:

[0120] Table 1. In vitro antitumor cell proliferation activity of the compounds

[0121]

[0122] As can be seen from the table, all tested compounds exhibit significant anti-cell proliferation activity.

[0123] Effects of compounds (1S,3R)-Ab and (1S,3R)-Bb on the proliferation of HT1080 cells in Test Example 2

[0124] The in vitro proliferative activity of compounds (1S,3R)-Ab and (1S,3R)-Bb against HT1080 cells was evaluated using the MTT assay, with RSL3 as a positive control. The IC50 values ​​of the compounds at 24 h and 48 h were analyzed using GraphPad Prism 8.3.0 software. 50 Values ​​(3 replicate experiments, data presented as: mean ± standard deviation)

[0125] The experimental results are shown in Table 2:

[0126] Table 2. In vitro antitumor cell proliferation activity of compounds (1S,3R)-Ab and (1S,3R)-Bb

[0127]

[0128] The results showed that compounds (1S,3R)-Ab and (1S,3R)-Bb reduced the IC50 of HT1080 cells by 24 h and 48 h. 50 The values ​​showed no difference, providing guidance for the action time in subsequent experiments.

[0129] Effects of compounds (1S,3R)-Ab and (1S,3R)-Bb on proliferation and ferroptosis selectivity of HT1080 cells in Test Example 3

[0130] Given the sensitivity of ferroptosis in HT1080 cells, the compounds (1S,3R)-Ab and (1S,3R)-Bb, which exhibited relatively good activity, were selected. Their cytotoxicity, used alone or in combination with the ferroptosis inhibitor fer-1 (1.0 μM), was tested. The IC50 values ​​of the combinations with the individual compounds were also assessed. 50 The ratio was used to assess their selectivity for ferroptosis. The specific method was as follows: HT1080 cells were cultured at 4 × 10⁻⁶... 4 Cells were seeded at a density of 10 cells / mL in 96-well plates. After attachment (approximately 6 hours), fresh culture medium containing different concentrations of the compound was added to each well. Simultaneously, fer-1 (final concentration 1.0 μM) was added, and after co-incubation for 48 hours, cell viability was assessed using the MTT assay as described above, and data were analyzed using GraphPad Prism 8.3.0 software.

[0131] The results are shown in Table 3 below:

[0132] Table 3. In vitro antitumor cell proliferation activity of compounds (1S,3R)-Ab and (1S,3R)-Bb

[0133]

[0134] Note: n=3; a HT1080: Cells and compounds are incubated separately; b HT1080: Cells were co-incubated with the compound and fer-1 (1.0 μM). c HT1080 b IC 50 / HT1080 a IC 50 .

[0135] The results showed that the IC50 values ​​of compounds (1S,3R)-Ab and (1S,3R)-Bb were different before and after the addition of fer-1. 50 The values ​​increased by 70-fold and 41.5-fold, respectively, suggesting that the inhibition of HT1080 cell proliferation may be achieved by inducing ferroptosis.

[0136] Effects of compounds (1S,3R)-Ab and (1S,3R)-Bb on GPX4 enzyme activity in Test Example 4

[0137] Main instruments and reagents

[0138] (1) Microplate reader (SpectraMax iD5): Meigu Molecular Instruments; Plate shaker (Titramax 100): Heidolph Corporation; Vortex shaker (VXMNDG): Ohaus Instruments (Shanghai) Co., Ltd.

[0139] (2) GPX4 Inhibitor Screening Assay Kit (Cayman, 701880-96wells)

[0140] Experimental methods

[0141] Assay Principle: This assay indirectly measures GPX4 through a coupling reaction with glutathione reductase (GR). Oxidized glutathione (GSSG) is produced by the reduction of hydrogen peroxide by GPX4, cycling to its reduced state via GR and NADPH, where NADPH is oxidized to NADP. + With the decrease in absorbance at 340 nm (A 340 ), A 340 The rate of decrease was directly proportional to GPX4 activity, with the GPX4 inhibitor ML162 used as a positive control.

[0142]

[0143]

[0144] First, according to the kit instructions, prepare all necessary reagents and buffers. Incubate the test compounds (all at 1 μM) dissolved in the GPX4 assay buffer with the GPX4 enzyme at room temperature for 1 hour. Then, according to the GPX4 inhibitor screening assay kit (Cayman, 701880-96wells) instructions, add a mixture of glutathione and glutathione reductase. Next, add NADPH and isopropyl hydrogen peroxide and mix thoroughly. Finally, perform kinetic detection using a microplate reader, measuring the absorbance at 340 nm every 30 seconds for 5 minutes. Process the data according to the instructions.

[0145] Experimental results

[0146] like Figure 2 As shown in Table 4, at a concentration of 1 μM, both compounds (1S,3R)-Ab and (1S,3R)-Bb inhibited GPX4 enzyme activity compared to 100% primordial enzyme activity, with inhibition rates of 45.14% and 41.84%, respectively, indicating that the compounds may induce ferroptosis in HT1080 cells by inhibiting GPX4 enzyme.

[0147] Table 4. Effects of compounds (1S,3R)-Ab and (1S,3R)-Bb on GPX4 enzyme activity.

[0148]

[0149] Note: n = 3, values ​​are expressed as Mean ± SD for each compound.

[0150] Effects of compounds (1S,3R)-Ab and (1S,3R)-Bb on intracellular reactive oxygen species (ROS) levels in HT1080 cells (Example 5)

[0151] In living organisms, the main sources of reactive oxygen species (ROS) are the mitochondrial inner membrane respiratory chain and NADPH oxidase. When the redox reaction in cells is imbalanced, ROS accumulates, causing lipid peroxidation of polyunsaturated fatty acids (PUFAs) in organelles and on the cell membrane, leading to the accumulation of lipid peroxides and inducing ferroptosis. To verify whether compounds (1S,3R)-Ab and (1S,3R)-Bb can induce ferroptosis by increasing intracellular ROS levels, we stained cells with the DCFH-DA probe and then detected intracellular ROS levels using flow cytometry.

[0152] Main instruments and reagents

[0153] (1) Flow cytometer: BD LSRF Ortessa custom flow cytometer

[0154] (2) DCFH-DA probe: Aladdin Reagent Company

[0155] Experimental methods

[0156] Measurement Principle: The fluorescent probe DCFH-DA is used to detect intracellular reactive oxygen species (ROS). DCFH-DA itself is non-fluorescent and can freely cross the cell membrane. Once inside the cell, it is hydrolyzed by intracellular esterases to generate DCFH. DCFH cannot cross the cell membrane, allowing the probe to be easily labeled intracellularly. In the presence of ROS, DCFH is oxidized to generate the green fluorescent substance DCF. The fluorescence intensity of DCF is directly proportional to the intracellular ROS level; therefore, detecting the fluorescence intensity of DCF reflects the intracellular ROS level.

[0157] The specific experimental procedure is as follows: HT1080 cells were cultured at a rate of 3 × 10⁻⁶. 5Cells were seeded at a density of 3 mL / well in 6-well plates. After 24 h of adherent growth, the cell culture medium was replaced with fresh culture medium containing (1S,3R)-Ab and (1S,3R)-Bb or RSL3 (positive control) at a concentration of 0.2 μM. After 12 h of incubation, the original drug-containing culture medium was discarded, and the cells were washed three times with PBS. Then, the cells were incubated for 40 min with serum-free culture medium containing the DCFH-DA probe (final concentration 10 μM). The culture medium was then discarded, and the cells were washed three times with PBS, digested with trypsin, and collected into 1.5 mL centrifuge tubes. Clean PBS was added to the centrifuge tubes, and the cells were centrifuged for 5 min (10000 rpm) after refluxing. This centrifugation was repeated three times to wash away any remaining probe that had not yet entered the cells. After centrifugation, 0.5 mL of clean PBS was added to the centrifuge tubes to prepare a homogenized cell solution. The solution was filtered through a nylon mesh and analyzed by flow cytometry (FCM).

[0158] Experimental results

[0159] like Figure 3 As shown, when the drug concentration was 0.2 μM, the compound (1S,3R)-Ab significantly increased the reactive oxygen species (ROS) level in HT1080 cells compared with the negative control group, and was comparable to the RSL3 level in the positive control group. However, there was no significant difference in ROS level between the compound (1S,3R)-Bb group and the negative control group, indicating that the compound (1S,3R)-Ab was more effective and may be a potential ferroptosis inducer.

[0160] Effect of compound (1S,3R)-Ab on malondialdehyde content in HT1080 cells (Example 6)

[0161] Malondialdehyde (MDA) is a product of lipid peroxidation, and its content can represent the degree of lipid peroxidation damage and is positively correlated with ferroptosis.

[0162] Main instruments and reagents

[0163] (1) Benchtop refrigerated centrifuge (5810r): eppendorf; Metal bath (ThermoStat C): eppendorf; Microplate reader (SpectraMax iD5): Meigu Molecular Instruments; Plate shaker (Titramax 100): Heidolph; Vortex shaker (VXMNDG): Ohaus Instruments (Shanghai) Co., Ltd.

[0164] (2) Lipid oxidation (MDA) assay kit (Beyotime, S0131S), Bradford protein concentration assay kit (detergent compatible) (Beyotime, P0006C).

[0165] Experimental methods

[0166] Determination principle: Malondialdehyde can react with TBA at higher temperatures and in acidic environments to form a red MDA-TBA adduct, which has maximum absorption at 535 nm, and can be detected by colorimetry.

[0167] The specific experimental procedure is as follows: HT1080 cells were seeded at a density of 3×10⁵ cells / mL into 6-well plates (3 mL per well). After 24 h of adherent growth, the cell culture medium was replaced with fresh culture medium containing (1S,3R)-Ab and RSL3 (positive control), with a drug concentration of 0.2 μM. After incubation for 24 h, the 6-well plates were placed on ice, the original drug-containing culture medium was discarded, and the cells were washed 3 times with PBS. Then, lysis buffer was added and the cells were lysed on ice for 5 min. The protein was harvested with a scraper, centrifuged at 13000 rpm for 15 min at 4 °C, and the supernatant was collected. The protein concentration was determined using a kit, and the malondialdehyde (MDA) content in the cells was determined according to the instructions. Finally, the MDA content per unit weight of protein was calculated.

[0168] Experimental results

[0169] like Figure 4 As shown, at a drug concentration of 0.2 μM, compound (1S,3R)-Ab significantly increased malondialdehyde levels in HT1080 cells compared to the negative control group, and was more effective than the positive control group RSL3, suggesting it may be a potential ferroptosis inducer.

[0170] Effect of compound (1S,3R)-Ab on GPX4 enzyme content in HT1080 cells (Test Example 7)

[0171] Ferroprelation is driven by lipid peroxidation and regulated at multiple levels. Generally, it is characterized by the accumulation of lipid peroxides in cells and the inability to utilize internal defense systems that should eliminate these peroxides. This leads to the accumulation of peroxides to lethal levels, damaging the phospholipids that make up the cell membrane and ultimately causing cell death. Yang et al. first identified GPX4 as a key regulator of ferroptosis in 2014. GPX4 is a selenoprotein and also a System xc - The core protein of the GSH-GPX4 axis (the main cellular system for defending against ferroptosis), which typically uses GSH as a cofactor, plays a key role in protecting cells from lipid peroxidation and ferroptosis by reducing lipid hydroperoxides to the corresponding lipid alcohols.

[0172] Main instruments and reagents

[0173] (1) Microplate reader (SpectraMax iD5): Meigu Molecular Instruments; Titramax 100 plate shaker: Heidolph; Vortex shaker (VXMNDG): Ohaus Instruments (Shanghai) Co., Ltd.; Metal bath (ThermoStatC): eppendorf; Chemdioc MP all-in-one chemiluminescence imager: Bio-Rad; Trans-Blot Turbo semi-dry transfer system: Bio-Rad; Mini-PROTEAN Tetra Cell vertical electrophoresis system: Bio-Rad

[0174] (2) Experimental methods

[0175] (1) Protein extraction: HT1080 cells were extracted at a concentration of 3 × 10⁻⁶. 5 The cells were seeded at a density of 3 mL / mL into 6-well plates. After 24 h of adherent growth, the cell culture medium was replaced with fresh culture medium containing (1S,3R)-Ab and RSL3 (positive control). After incubation for 24 h, the 6-well plates were placed on ice, the original drug-containing culture medium was discarded, and the cells were washed 3 times with PBS. Then, lysis buffer was added and the cells were lysed on ice for 5 min. The protein was harvested with a scraper, centrifuged at 13000 rpm for 15 min at 4 °C, and the supernatant was collected for subsequent protein concentration determination.

[0176] (2) Determination of protein concentration using BSA kit: After diluting the extracted protein, determine the protein content according to the kit instructions.

[0177] (3) Protein denaturation: After the protein concentration is determined, the mixture is leveled, and then 5× protein loading buffer is added. After vortexing and mixing evenly, the mixture is placed in a metal bath and boiled at 100°C for 15 minutes.

[0178] (4) Sample loading and electrophoresis: Fill the electrophoresis tank with electrophoresis buffer (Servicebio electrophoresis powder), use Servicebio's SDS-PAGE pre-cast gel, remove the comb, take a certain volume of the processed protein sample and marker, and load the sample in sequence. After loading, set the voltage to 100V and the time to 80 minutes.

[0179] (5) Transfer: Cut a PVDF membrane of appropriate size, activate it in methanol solution and put it into the transfer solution for later use. Then, use the semi-dry transfer method and transfer time of 5 min. It can be seen that the target protein on the gel has been completely transferred to the PVDF membrane.

[0180] (6) 5% skim milk sealing: After cutting the PVDF membrane with scissors, put it into an incubation box containing 5% skim milk, and then place it on a shaker and seal it at room temperature for 3 hours.

[0181] (7) Primary antibody incubation: After blocking the PVDF membrane, discard the skim milk and add an appropriate amount of TBST to wash the PVDF membrane for 10 minutes each time, for a total of 3 washes. After the last wash, cut the membrane according to the marker, and place the internal control (β-actin) and GPX4 enzyme in separate incubators and incubate overnight at 4°C.

[0182] (8) Incubation with secondary antibody: After the primary antibody incubation is completed, the primary antibody is recovered, and then an appropriate amount of TBST is added to wash the PVDF membrane for 10 minutes each time, for a total of 3 washes; after washing, the secondary antibody (prepared with 5% skim milk, diluted 1:2000) is added and incubated on a shaker at room temperature for 1 hour.

[0183] (9) Development: After the secondary antibody incubation is completed, discard the secondary antibody, then add an appropriate amount of TBST to wash the PVDF membrane for 10 min each time, for a total of 3 washes; after washing, prepare the developing solution (Ecl A solution and B solution, 1:1 mixture), then put the cut PVDF membrane together once, add 1 mL of developing solution to the membrane, and develop after 1-2 min.

[0184] Experimental results

[0185] like Figure 5 As shown, compared with the negative control group, compound (1S,3R)-Ab dose-dependently reduced the expression of GPX4 enzyme in HT1080 cells, suggesting that it may induce ferroptosis by inhibiting the activity of GPX4 enzyme in HT1080 cells.

[0186] Test Example 8: Evaluation of in vivo antitumor activity (transplanted BALB / c (♀) mouse 4T1 orthotopic tumor model)

[0187] laboratory animals

[0188] BALB / c mice (♀), SPF grade, weighing 18.0±1.0g, were purchased from Beijing Vital River Animal Experiment Technology Co., Ltd.

[0189] Grouping of experimental animals, administration method and dosage

[0190] (1) Experimental groups: normal group (Sham group, 3 animals), vehicle control (negative control solvent group), (1R,3S)-Ab group, (1S,3R)-Ab group, (1R,3R)-Ab group, (1S,3S)-Ab group, (1S,3R)-Bb group, (1S,3R)-Ac group, RSL3 (positive control group), except for the Sham group, 9 animals in each group, for a total of 75 animals.

[0191] (2) Administration method: Intraperitoneal injection (0.1 mL / 10 g), solvent: (2.5% DMSO + 10% castor oil + 20% PEG-400 + 67.5% pure water)

[0192] (3) Dosage: All were administered at a dose of 66 μmol / kg / day.

[0193] Construction of experimental animal models

[0194] Cells in the logarithmic growth phase were collected, digested with trypsin, and then washed three times with physiological saline by centrifugation. The cells were counted, and fresh physiological saline was added to prepare tumor fluid, with a seeding density of 1.0 × 10⁶ cells / year. 7 0.2 mL of tumor fluid per mouse was injected orally into the mammary gland of the fourth pair of mammary glands. When the tumor volume reached 100 mm², the tumor was treated. 3 Mice were randomly divided into groups and administered the drug for 13 consecutive days. Daily changes in body weight and tumor volume were recorded. The results were calculated using the equation V = L × W. 2 / 2 Calculate the tumor volume, where L represents the tumor length and W represents the tumor width; after 13 days of continuous administration, the mice were euthanized, and the mouse organs and tumors were collected for analysis. The tumor growth inhibition rate (TGI) was calculated as [1 - tumor weight of the administration group / tumor weight of the negative control group] × 100%.

[0195] Experimental Results and Discussion

[0196] like Figure 6 As shown in Table 5, compared with the negative control group, except for the compound (1R,3S)-Ab group, the tumor weight of the other treatment groups, namely the compound (1R,3S)-Ab group, (1S,3R)-Ab group, (1R,3R)-Ab group, (1S,3R)-Bb group, and (1S,3R)-Ac group, was reduced significantly, indicating that the designed compounds can delay the growth of 4T1 tumors in mice. In addition, the tumor inhibition rate of the compound (1S,3R)-Ab group and the (1S,3R)-Bb group was higher than that of the positive control drug RSL3, achieving unexpected results.

[0197] Table 5 Effects of compounds on mouse 4T1 orthotopic tumors

[0198]

[0199] Note: n=9, dosage 66μmol / kg, one-way ANOVA analysis was used.

[0200] a) Compared with Vehicle, p>0.05; b) Compared with Vehicle, p<0.05; c) Compared with Vehicle, p<0.01;

[0201] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An inhibitor targeting glutathione peroxidase 4 (GPX4), characterized in that... The inhibitor can selectively interfere with the activity of GPX4, and the structural formula of the inhibitor is as follows: In formula (I), R1 is any one of the substituents shown in A to F: R2 is any one of the substituents shown in b and c:

2. The inhibitor against GPX4 according to claim 1, characterized in that, The inhibitor is any one of the following compounds:

3. The method for preparing the inhibitor according to claim 1 or 2, comprising the following steps: (1) In the presence of thionyl chloride, L-tryptophan / D-tryptophan reacts with methanol to form the compound L / D-tryptophan methyl ester hydrochloride (1-S,1-R). (2) Under the catalysis of concentrated sulfuric acid, L / D-tryptophan methyl ester hydrochloride and different aromatic aldehydes (caminaldehyde, methyl 5-formyl salicylate, eugenol, vanillin, cinnamaldehyde) were heated under reflux in methanol solution to carry out Pictet-Spengler condensation reaction to obtain compounds (2-SS, 2-RS, 2-RR, 2-SR). (3) When the R2 substituent is b or c, the intermediate obtained in step (2) is dissolved in dry dichloromethane, and an appropriate amount of triethylamine or sodium bicarbonate solid is added to the solution. At 0°C, the acyl chlorides corresponding to b and c (b, chloroacetyl chloride; c, acryloyl chloride) are added dropwise to finally obtain the compound (3-SS, 3-RS, 3-RR, 3-SR).

4. Use of the compound of claim 1, its pharmaceutically acceptable salt, N-oxide, hydrate, solvate, metabolite, polymorph or prodrug, or tautomer, meso compound, racemic, enantiomer, diastereomer, or mixture thereof, in the preparation of a GPX4 protein inhibitor or in the treatment of cancer, wherein the cancer is diffuse large B-cell lymphoma, oral squamous cell carcinoma, glioma, lung cancer, colorectal cancer, bladder cancer, gastric cancer, urothelial carcinoma, prostate cancer, breast cancer, liver cancer, cervical cancer, or thyroid cancer.

5. As described in claim 4, characterized in that The tumor is a tumor that highly expresses GPX4.

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