Polyhydroxy-substituted benzophenone derivatives and their application in preventing hepatocyte ferroptosis

Abstract

This invention relates to the field of pharmaceutical technology, and particularly to polyhydroxy-substituted benzophenone derivatives and their application in preventing hepatocyte ferroptosis. The polyhydroxy-substituted benzophenone derivatives have the following structural formula: [structural formula would be inserted here], where R1 = ethyl, n-propyl, isopropyl; R2 = hydrogen, bromine, chlorine; and R3 = butyryloxy, morpholine-4-carbonyloxy, hydroxyl. The polyhydroxy-substituted benzophenone derivatives of this invention exhibit significant effects in protecting hepatocytes by inhibiting ferroptosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to polyhydroxy-substituted benzophenone derivatives and their application in preventing hepatocyte ferroptosis. Background Technology

[0002] Benzophenones were initially discovered in natural products and are widely found in higher plants, such as those in the Agaricus, Iris, and Rosaceae families. They are also widely distributed in marine organisms, such as marine fungi, algae, and mollusks. Benzophenones have been shown to possess a wide range of biological activities, including anticancer, anti-inflammatory, antibacterial, antiviral, protein kinase inhibitory, and sulfatase inhibitory activities. Benzophenone, as a widely found chemical skeleton with diverse biological activities, has been used in various drugs. Marketed drugs containing the benzophenone skeleton include the nonsteroidal anti-inflammatory drug ketoprofen, tocapone for treating Parkinson's disease, and fenofibrate for treating hypercholesterolemia. The effects of these drugs primarily focus on anti-inflammatory, α-glucosidase inhibitory, and anti-atherosclerotic effects.

[0003] Ferroprelation is a novel form of programmed cell death, distinct from apoptosis, classical necrosis, autophagy, and other forms of cell death. Its triggering is associated with the inhibition of glutathione (GSH) biosynthesis, the inactivation of the glutathione-dependent antioxidant enzyme glutathione peroxidase 4 (GPX4), and the accumulation of iron-dependent reactive oxygen species (ROS). Its characteristics include alterations in mitochondrial number and the accumulation of lipotoxic substances such as MDA. Current research demonstrates that ferroptosis plays a crucial role in drug-induced liver injury, ischemia-reperfusion liver injury, acute or chronic liver diseases, and hepatocellular carcinoma. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a polyhydroxy-substituted benzophenone derivative and its application in preventing hepatocyte ferroptosis.

[0005] The technical solution adopted in this invention is: a polyhydroxy-substituted benzophenone derivative, characterized by the following structural formula: Where R1 = hydrogen, methyl, ethyl, n-propyl, isopropyl, R2 = hydrogen, bromine, chlorine, and R3 = butyryloxy, morpholine-4-carbonyloxy, hydroxyl.

[0006] The aforementioned polyhydroxy-substituted benzophenone derivatives are characterized by the following structural formula: Where R1 = hydrogen, methyl, ethyl, n-propyl, isopropyl, R2 = hydrogen, bromine, chlorine, and R3 = hydroxyl.

[0007] The aforementioned polyhydroxy-substituted benzophenone derivatives are characterized by the following structural formula: Where R1 = hydrogen, methyl, ethyl, n-propyl, isopropyl, R2 = hydrogen, and R3 = butyryloxy, morpholine-4-carbonyloxy, hydroxyl.

[0008] The application of polyhydroxy-substituted benzophenone derivatives in preventing hepatocyte ferroptosis; the structural formula of polyhydroxy-substituted benzophenone derivatives is as follows: Where R1 = hydrogen, methyl, ethyl, n-propyl, isopropyl, R2 = hydrogen, bromine, chlorine, and R3 = butyryloxy, morpholine-4-carbonyloxy, hydroxyl.

[0009] The application of the aforementioned polyhydroxy-substituted benzophenone derivatives in preventing hepatocyte ferroptosis is characterized by the following structural formula: Where R1 = hydrogen, methyl, ethyl, n-propyl, isopropyl, R2 = hydrogen, bromine, chlorine, and R3 = hydroxyl.

[0010] The application of the aforementioned polyhydroxy-substituted benzophenone derivatives in preventing hepatocyte ferroptosis is characterized by the following structural formula: .

[0011] The application of the aforementioned polyhydroxy-substituted benzophenone derivatives in preventing hepatocyte ferroptosis is characterized by the following structural formula: Where R1 = hydrogen, methyl, ethyl, n-propyl, isopropyl, R2 = hydrogen, and R3 = butyryloxy, morpholine-4-carbonyloxy, hydroxyl.

[0012] The application of the aforementioned polyhydroxy-substituted benzophenone derivatives in preventing hepatocyte ferroptosis is characterized by the following structural formula: .

[0013] The application of the aforementioned polyhydroxy-substituted benzophenone derivatives in preventing hepatocyte ferroptosis is characterized by the following structural formula: .

[0014] This invention synthesizes a series of polyhydroxy-substituted benzophenone derivatives through the preparation of acyl chlorides, Friedel-Crafts acylation, halogenation on the aromatic ring, demethylation of boron tribromide, and esterification, and verifies their application in protecting hepatocytes by inhibiting ferroptosis. Attached Figure Description

[0015] Figure 1 Characterization table of compounds 1-5 of this invention;

[0016] Figure 2 Characterization table of compounds 6-10 of this invention;

[0017] Figure 3Characteristic table of compounds 11-14 of the present invention;

[0018] Figure 4 Characterization table of compounds 15-18 of this invention;

[0019] Figure 5 Compounds 1-18 of this invention are in 25 µmol·L⁻¹ -1 Results of toxicity to hepatocytes;

[0020] Figure 6 Comparison of iron ion content in cells treated with ferroptosis inducer Erastin and cells treated with ferroptosis inducer Erastin + fenofibrate, ferroptosis inducer Erastin + compound 5, ferroptosis inducer Erastin + compound 13, and ferroptosis inducer Erastin + compound 18.

[0021] Figure 7 Comparison of ROS content in cells treated with ferroptosis inducer Erastin and cells treated with ferroptosis inducer Erastin + fenofibrate, ferroptosis inducer Erastin + compound 5, ferroptosis inducer Erastin + compound 13, and ferroptosis inducer Erastin + compound 18.

[0022] Figure 8 Comparison of MDA content in hepatocyte culture supernatant after treatment with ferroptosis inducer Erastin and hepatocyte culture supernatant after treatment with ferroptosis inducers Erastin + fenofibrate, Erastin + compound 5, Erastin + compound 13, and Erastin + compound 18.

[0023] Figure 9 A comparison of ALT content in hepatocyte culture supernatant after treatment with the ferroptosis inducer Erastin and after treatment with the ferroptosis inducers Erastin + fenofibrate, Erastin + compound 5, Erastin + compound 13, and Erastin + compound 18.

[0024] Figure 10 Comparison of AST content in hepatocyte culture supernatant after treatment with the death inducer Erastin and the hepatocyte culture supernatant after treatment with the ferroptosis inducers Erastin + fenofibrate, Erastin + compound 5, Erastin + compound 13, and Erastin + compound 18.

[0025] Figure 11Comparison of LDH content in hepatocyte culture supernatant treated with Erastin (a ferroptosis inducer) and hepatocyte culture supernatant treated with Erastin (a ferroptosis inducer) + fenofibrate, Erastin (a ferroptosis inducer) + compound 5, Erastin (a ferroptosis inducer) + compound 13, and Erastin (a ferroptosis inducer) + compound 18.

[0026] Figure 12 Comparison of mitochondrial numbers in cells treated with ferroptosis inducer Erastin and cells treated with ferroptosis inducer Erastin + fenofibrate, ferroptosis inducer Erastin + compound 5, ferroptosis inducer Erastin + compound 13, and ferroptosis inducer Erastin + compound 18.

[0027] Figure 13 Comparison of GSH content in hepatocyte culture supernatant after treatment with the death inducer Erastin and the hepatocyte culture supernatant after treatment with the ferroptosis inducers Erastin + fenofibrate, Erastin + compound 5, Erastin + compound 13, and Erastin + compound 18.

[0028] Figure 14 Comparison of morphological changes in cells treated with ferroptosis inducer Erastin with those treated with Erastin + fenofibrate, Erastin + compound 5, Erastin + compound 13, and Erastin + compound 18. Detailed Implementation

[0029] A polyhydroxy ketone derivative is prepared by the following method:

[0030] (1) Synthesis of acyl chloride

[0031] Benzoic acid, methylbenzoic acid, ethylbenzoic acid, n-propylbenzoic acid, isopropylbenzoic acid, 4-isopropoxybenzoic acid, and 4-propoxybenzoic acid were dissolved in SOCl2, DMF was added, and the mixture was heated to reflux. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, and an appropriate amount of anhydrous CH2Cl2 was added. Excess SOCl2 was removed by vacuum distillation. Benzoyl chloride, p-methylbenzoyl chloride, p-ethylbenzoyl chloride, p-n-propylbenzoyl chloride, p-isopropylbenzoyl chloride, p-isooxypropylbenzoyl chloride, and p-propoxybenzoyl chloride were obtained. The reaction formula is as follows:

[0032]

[0033] In the formula, R1 = H, methyl, ethyl, n-propyl, isopropyl, isopropoxy, propoxy.

[0034] (2) Friedel-Crafts acylation reaction

[0035] Phthalic dimethyl ether or 1,2,3-trimethoxybenzene was dissolved in anhydrous CH2Cl2, and crude acyl chloride was dissolved in an appropriate amount of anhydrous CH2Cl2. The solutions were slowly added dropwise to the reaction system. During the reaction, anhydrous aluminum chloride (AlCl3) was weighed and added to the reaction solution in three portions, and the reaction progress was monitored by TLC. After the reaction was completed, ice water was added to quench the reaction. The reaction solution was extracted with CH2Cl2, the organic phases were combined, dried over anhydrous sodium sulfate for 2 h, the solvent was removed by silica gel rotation, and the product was purified by silica gel column chromatography to obtain the Friedel-Crafts acylated products. The reaction formulas for phenyl(2,3,4-trimethoxyphenyl) ketone, p-tolyl(2,3,4-trimethoxyphenyl) ketone, (4-ethylphenyl)(2,3,4-trimethoxyphenyl) ketone, (4-propylphenyl)(2,3,4-trimethoxyphenyl) ketone, and (4-isopropylphenyl)(2,3,4-trimethoxyphenyl) ketone are as follows:

[0036]

[0037] In the formula, R1 = hydrogen, methyl, ethyl, n-propyl, isopropyl.

[0038] (3) Halogenation reaction

[0039] ① Chlorination

[0040] The synthesized product was dissolved in an appropriate amount of anhydrous CH2Cl2. SO2Cl2 was slowly added dropwise to the reaction system. The reaction progress was monitored by TLC. After the reaction was completed, the product was extracted with anhydrous dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate for 2 h, and the solvent was removed by silica gel electrophoresis. The product was then purified by silica gel column chromatography to obtain (6-chloro-2,3,4-trimethoxyphenyl)(phenyl) methyl ketone, (6-chloro-2,3,4-trimethoxyphenyl)(p-tolyl) methyl ketone, (6-chloro-2,3,4-trimethoxyphenyl)(4-ethylphenyl) methyl ketone, (6-chloro-2,3,4-trimethoxyphenyl)(4-propylphenyl) methyl ketone, and (6-chloro-2,3,4-trimethoxyphenyl)(4-isopropylphenyl) methyl ketone. The reaction formula is as follows:

[0041]

[0042] In the formula, R1 = hydrogen, methyl, ethyl, n-propyl, isopropyl.

[0043] ② Brominated

[0044] The Friedel-Crafts acylation product was dissolved in an appropriate amount of anhydrous CH2Cl2. N-bromosuccinimide (NBS) was slowly added dropwise to the reaction system. The reaction progress was monitored by TLC. After the reaction was complete, the mixture was extracted with anhydrous dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate for 2 h, and the solvent was removed by silica gel rotation. The mixture was then purified by silica gel column chromatography to obtain (6-bromo-2,3,4-trimethoxyphenyl)(phenyl) ketone, (6-bromo-2,3,4-trimethoxyphenyl)(p-tolyl) ketone, (6-bromo-2,3,4-dimethoxyphenyl)(4-ethylphenyl) ketone, (6-bromo-2,3,4-trimethoxyphenyl)(4-propylphenyl) ketone, and (6-bromo-2,3,4-trimethoxyphenyl)(4-isopropylphenyl) ketone. The reaction formula is as follows:

[0045]

[0046] In the formula, R1 = hydrogen, methyl, ethyl, n-propyl, isopropyl.

[0047] (4) Demethylation reaction

[0048] The above Friedel-Crafts acylation product or halogenation product compound was dissolved in 15 mL of anhydrous CH2Cl2. BBr3 was slowly added dropwise to the reaction system. The reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was slowly added dropwise to 10 mL of ice water to quench the reaction. The reaction solution was extracted with CH2Cl2, the organic phases were combined, dried over anhydrous sodium sulfate for 2 h, the solvent was removed by silica gel rotation, and the mixture was purified by silica gel column chromatography. The resulting compounds reacted with the Friedel-Crafts acylation product to give phenyl(2,3,4-trihydroxyphenyl) methyl ketone, p-tolyl(2,3,4-trihydroxyphenyl) methyl ketone, (4-ethylphenyl)(2,3,4-trihydroxyphenyl) methyl ketone, (4-propylphenyl)(2,3,4-trihydroxyphenyl) methyl ketone, and (4-isopropylphenyl)(2,3,4-trihydroxyphenyl) methyl ketone. The reaction formula is as follows:

[0049]

[0050] In the formula, R1 = hydrogen, methyl, ethyl, n-propyl, isopropyl, and R2 = hydrogen, bromine, chlorine.

[0051] The product of the halogenation reaction yields (6-chloro-2,3,4-trihydroxyphenyl)(phenyl) methyl ketone, (6-chloro-2,3,4-trihydroxyphenyl)(p-tolyl) methyl ketone, (6-chloro-2,3,4-trihydroxyphenyl)(4-ethylphenyl) methyl ketone, (6-chloro-2,3,4-trihydroxyphenyl)(4-propylphenyl) methyl ketone, (6-chloro-2,3,4-dihydroxyphenyl)(4-isopropylphenyl) methyl ketone, (6-bromo-2,3,4-trihydroxyphenyl)(phenyl) methyl ketone, (6-bromo-2,3,4-trihydroxyphenyl)(p-tolyl) methyl ketone, (6-bromo-2,3,4-trihydroxyphenyl)(4-propylphenyl) methyl ketone, and (6-bromo-2,3,4-trihydroxyphenyl)(4-isopropylphenyl) methyl ketone.

[0052] (5) Esterification reaction

[0053] The Friedel-Crafts acylation and demethylation product from the previous step was dissolved in 15 mL of anhydrous CH2Cl2. Pyridine was added as a catalyst, followed by n-butyryl chloride or 4-morpholine carbamate chloride. The reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was extracted with CH2Cl2, and the organic phases were combined and dried over anhydrous sodium sulfate for 2 hours. h, solvent was removed by silica gel rotation, and the mixture was purified by silica gel column chromatography to give compounds 4-benzoyl-2,3-dihydroxyphenylbutyrate, 4-benzoyl-2,3-dihydroxyphenylmorpholine-4-carboxylate, 2,3-dihydroxy-4-(4-methylbenzoyl)phenylbutyrate, 2,3-dihydroxy-4-(4-methylbenzoyl)phenylmorpholine-4-carboxylate, 4-(4-ethylbenzoyl)-2,3-dihydroxyphenylbutyrate, 4-(4-ethylbenzoyl)-2,3-dihydroxyphenylmorpholine-4-carboxylate, 2,3-dihydroxy-4-(4-propylbenzoyl)phenylbutyrate, 2,3-dihydroxy-4-(4-propylbenzoyl)phenylmorpholine-4-carboxylate, and 2,3-dihydroxy-4-(4-isopropylbenzyl)phenylbutyrate. The reaction formula is as follows:

[0054]

[0055] In the formula, R1 = hydrogen, methyl, ethyl, n-propyl, isopropyl, and R3 = butyryloxy, morpholine-4-carbonyloxy, hydroxyl.

[0056] The compounds prepared above are as follows Figure 1-4 As shown.

[0057] The structures of each compound are characterized as follows:

[0058] 4-Benzoyl-2,3-dihydroxyphenylbutyrate (Compound 1)

[0059] Molecular formula: C 17 H16 O5; Yellow solid , Yield: 40.1%; mp: 85.7-87.2℃; 1 H NMR (600 MHz, CDCl3) δ 12.74 (s, 1H), 7.60 (d, J = 7.3 Hz, 2H), 7.55 (t,J = 7.2 Hz, 1H), 7.47 (t, J =7.5 Hz, 2H), 7.36 (d, J = 8.9 Hz, 1H), 6.45 (d,J = 8.9 Hz, 1H), 2.66 (t, J = 7.3 Hz, 2H), 1.83 (dt, J = 14.8, 7.4 Hz, 2H), 1.07 (t, J = 7.3 Hz, 3H). 13 C NMR (151 MHz, CDCl3) δ = 200.36, 171.39, 157.33,154.86, 137.90, 132.15, 131.67, 129.15, 128.89, 128.33,113.82, 107.46, 77.24,77.03, 76.81, 35.69, 18.53, 13.56. m / z(-ESI): 301.14 [M+H] - .

[0060] 4-Benzoyl-2,3-dihydroxyphenylmorpholine-4-carboxylate (Compound 2)

[0061] Molecular formula: C18H17NO6; Yellow solid , Yield: 42.3%; mp: 228.2-228.7 ℃; 1H NMR (600 MHz, CDCl3) δ 12.80 (s, 1H), 7.62 (d, J = 7.1 Hz, 2H),7.58 – 7.55 (m, 1H), 7.49 (t, J = 7.6 Hz, 2H), 7.38 (d, J = 9.0Hz, 1H), 6.50(d, J = 9.0 Hz, 1H), 3.86 (d, J = 3.1 Hz, 2H), 3.82 – 3.77 (m, 4H), 3.63 (s,2H). 13C NMR (151 MHz, CDCl3) δ = 200.36, 157.53, 155.26, 153.86, 137.91,131.90, 131.74, 128.90, 128.37, 128.13, 113.78, 108.64, 77.22, 77.01, 76.80,66.49,45.57. m / z(-ESI): 344.17 [M+H]-.

[0062] (6-Bromo-2,3,4-Trihydroxyphenyl)(phenyl)methyl ketone (Compound 3)

[0063] Molecular formula: C13H9BrO4; Yellow solid, Yield: 53.2%; mp: 125.7-126.2 ℃; 1H NMR (600 MHz, CDCl3) δ 12.51 (s, 1H), 7.66 (d, J = 7.2 Hz, 2H),7.62 (t, J = 7.5 Hz, 1H), 7.53 (t, J =7.6 Hz, 2H), 7.39 (s, 1H). 13C NMR (151MHz, CDCl3) δ = 198.91, 149.67, 145.64, 136.29, 131.33, 131.16, 127.96,127.54, 127.02, 113.01, 98.46, 76.21, 76.00, 75.79. m / z(-ESI): 309.01[M+H]-.

[0064] (6-Chloro-2,3,4-Trihydroxyphenyl)(phenyl)methyl ketone (Compound 4)

[0065] Molecular formula: C13H9ClO4; Yellow solid, Yield: 37.9%; mp: 112.7-114.2 ℃; 1H NMR (600 MHz, CDCl3) δ 12.51 (s, 1H), 7.66 (d, J = 7.1 Hz, 2H),7.61 (t, J = 7.5 Hz, 1H), 7.53 (t, J =7.6 Hz, 2H), 7.24 (s, 1H). 13C NMR (151MHz, CDCl3) δ = 198.87, 149.67, 145.64, 136.29, 131.33, 131.16, 127.96,127.54, 127.02, 113.01, 98.46, 76.21, 76.00, 75.79. m / z(-ESI): 265.04[M+H]-.

[0066] 2,3-Dihydroxy-4-(4-methylbenzoyl)phenylbutyrate (compound 5)

[0067] Molecular formula: C18H18O5; White solid, Yield: 37.6%; mp: 104.8-105.2 ℃; 1H NMR (600 MHz, CDCl3) δ 12.79 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H),7.40 (d, J = 8.9 Hz, 1H), 7.29 (d, J =9.4 Hz, 1H), 7.26 (d, J = 4.6 Hz, 1H), 6.44 (d, J = 8.9 Hz, 1H), 2.66 (t, J = 7.4 Hz, 2H), 2.43 (s, 3H), 1.87 – 1.80(m, 2H), 1.07 (t, J = 7.4 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ = 200.17,171.38, 157.16, 154.40, 142.49, 135.09, 132.10, 129.46, 129.19, 129.10,129.02, 107.24, 77.25, 77.04, 76.82, 35.69,21.58, 18.54, 13.61. m / z(-ESI):315.25 [M+H]-.

[0068] 2,3-Dihydroxy-4-(4-methylbenzoyl)phenylmorpholine-4-carboxylate (Compound 6)

[0069] Molecular formula: C19H19NO6; Yellow solid, Yield: 42.6%; mp: 206.9-207.1 ℃; 1H NMR (600 MHz, CDCl3) δ 12.82 (s, 1H), 7.51 (d, J = 7.7 Hz, 2H), 7.35 (d, J =8.9 Hz, 1H), 7.26 (d, J = 7.2 Hz, 2H), 6.42 (d, J = 8.9 Hz, 1H), 3.80 (d, J = 20.9 Hz, 4H), 3.78 – 3.57 (m, 4H), 2.43 (s, 3H). 13C NMR (151MHz, CDCl3) δ = 200.06, 157.50, 155.38, 153.91, 142.47, 135.13, 131.77,129.18, 129.01, 127.73,113.72, 108.23, 77.25, 77.04, 76.83, 66.48, 45.49,44.61, 29.32, 21.58. m / z(-ESI): 358.28 [M+H]-.

[0070] (6-Bromo-2,3,4-Trihydroxyphenyl)(p-Tolyl)methyl ketone (Compound 7)

[0071] Molecular formula: C14H11BrO4; Yellow solid, Yield: 45.1%; mp: 155.2-155.7 ℃; 1H NMR (600 MHz, DMSO) δ 11.80 (s, 1H), 7.56 (d, J = 7.9 Hz, 2H), 7.37 (d, J =7.8 Hz, 2H), 7.13 (s, 1H), 2.41 (s, 3H). 13C NMR (151 MHz, DMSO)δ = 198.65, 151.29, 149.88, 134.45, 129.79, 129.57, 129.49, 129.43, 126.96,114.64, 100.50, 40.46, 40.32, 40.18, 40.05, 39.91,39.77, 39.63, 21.58. m / z(-ESI): 322.93 [M+H]-.

[0072] (6-Chloro-2,3,4-Trihydroxyphenyl)(p-Tolyl)methyl ketone (Compound 8)

[0073] Molecular formula: C14H11ClO4; Yellow solid, Yield: 39.3%; mp: 150.2-150.8 ℃; 1H NMR (600 MHz, DMSO) δ 11.75 (s, 1H), 10.50 (s, 1H), 9.47 (s,1H), 7.60 (d, J =38.4 Hz, 2H), 7.34 (d, J = 38.6 Hz, 2H), 6.98 (s, 1H), 2.41(s, 3H). 13C NMR (151 MHz, DMSO) δ = 198.67,150.75, 148.84, 142.80, 135.31,134.72, 129.80, 129.48, 123.85, 113.78, 111.94, 40.46, 40.32, 40.18, 40.04,39.91, 39.77, 39.63, 21.58. m / z(-ESI): 279.04[M+H]-.

[0074] 4-(4-Ethylbenzoyl)-2,3-dihydroxyphenylbutyrate (Compound 9)

[0075] Molecular formula: C19H20O5; Yellow liquid, Yield: 43.2%; mp: 133.7-135.2 ℃; 1H NMR (600 MHz, CDCl3) δ 12.44 (s, 1H), 7.62 (d, J = 6.6 Hz, 2H), 7.55 (d, J = 8.2 Hz, 1H), 7.33 (d, J =6.4 Hz, 2H), 6.73 (d, J = 8.2 Hz, 1H), 2.73 (t, J = 17.3 Hz, 2H), 2.58 (d, J = 37.9 Hz, 2H), 1.83 – 1.78 (m, 2H), 1.29 – 1.26 (m, 3H), 1.08 – 1.04 (m, 3H). 13C NMR (151 MHz, CDCl3) δ =200.55, 170.29, 156.95, 149.26, 148.44, 134.99, 131.69, 130.83, 129.52,127.98, 118.13, 112.85, 77.24, 77.03, 76.82, 35.96,35.60, 28.95, 18.55,18.39. m / z(-ESI): 329.27 [M+H]-.

[0076] 4-(4-Ethylbenzoyl)-2,3-Dihydroxyphenylmorpholine-4-carboxylate (Compound 10)

[0077] Molecular formula: C20H21NO6; White solid, Yield: 34,7%; mp: 193.8-194.2 ℃; 1H NMR (600 MHz, CDCl3) δ 12.83 (s, 1H), 7.53 (d, J = 8.1 Hz, 2H),7.37 (d, J = 9.0 Hz, 1H), 7.29 (d, J =8.1 Hz, 2H), 6.43 (d, J = 9.0 Hz, 1H), 3.83 – 3.80 (m, 2H), 3.80 – 3.69 (m, 4H), 3.63 – 3.59 (m, 2H), 2.73 (q, J =7.6 Hz, 2H), 1.28 (t, J = 7.6Hz, 3H). 13C NMR (151 MHz, CDCl3) δ = 200.08,171.19, 157.50, 155.34, 153.91, 148.67, 135.35, 131.81, 129.28, 127.85,127.77, 113.75, 108.23, 77.25, 77.03, 76.82, 66.49, 45.51, 44.44, 28.91,19.27, 15.37. m / z(-ESI): 372.29 [M+H]-.

[0078] (6-Chloro-2,3,4-Trihydroxyphenyl)(4-Ethylphenyl)methyl ketone (Compound 11)

[0079] Molecular formula: C15H13ClO4; Yellow solid, Yield: 35.7%; mp: 130.2-131.0 ℃; 1H NMR (600 MHz, CDCl3) δ 12.56 (s, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.34 (d, J =8.0 Hz, 2H), 7.27 (s, 1H), 2.75 (q, J = 7.6 Hz, 2H), 1.30 (t, J =7.6 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ = 199.80,150.24, 149.22, 145.73,134.80, 132.59, 129.35, 128.05, 125.03, 113.19, 111.43, 77.24, 77.03, 76.82,28.95, 15.21. m / z(-ESI): 293.14 [M+H]-.

[0080] 2,3-Dihydroxy-4-(4-propylbenzoyl)phenylbutyrate (Compound 12)

[0081] Molecular formula: C20H22O5; Yellow solid, Yield: 43.7%; mp: 143.2-143.7 ℃; 1H NMR (400 MHz, CDCl3) δ 12.80 (s, 1H), 7.53 (d, J = 8.1 Hz, 2H),7.41 (d, J = 8.9 Hz, 1H), 7.27 (d, J =8.1 Hz, 2H), 6.45 (d, J = 8.9 Hz, 1H), 2.67 (d, J = 7.3 Hz, 2H), 2.66 – 2.59 (m, 2H), 1.89 – 1.79 (m, 2H), 1.73 –1.63 (m, 2H), 1.07 (t, J = 7.4 Hz, 3H),0.96 (t, J = 7.3 Hz, 3H). 13C NMR (101MHz, CDCl3) δ = 200.22, 171.41, 157.17, 154.41, 147.21, 135.30, 132.16,129.49, 129.20, 128.45, 113.99, 107.24, 77.36, 77.05, 76.73, 38.01,35.69,24.30, 18.55, 13.81, 13.62. m / z(-ESI): 340.34 [M+H]-.

[0082] 2,3-Dihydroxy-4-(4-propylbenzoyl)phenylmorpholine-4-carboxylate (Compound 13)

[0083] Molecular formula: C21H23NO6; Yellow solid, Yield: 40.3%; mp: 143.7-144.2 ℃; 1H NMR (400 MHz, DMSO) δ 12.33 (s, 1H), 7.58 (d, J = 8.2 Hz, 2H),7.37 (d, J = 8.2 Hz, 2H), 7.32 (d, J =8.9 Hz, 1H), 6.53 (d, J = 8.9 Hz, 1H), 3.66 – 3.59 (m, 4H), 3.40 – 3.30 (m, 4H), 2.69 – 2.62 (m, 2H), 1.65 (dt, J =15.0, 7.4 Hz, 2H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO) δ =199.26, 157.24, 157.08, 152.78, 147.08, 135.72, 131.56, 129.46, 128.86,127.23, 113.37, 108.37, 68.74, 66.29, 40.63, 40.42, 40.21, 40.00, 39.79,39.58, 39.37,37.59, 31.77, 24.26, 14.23, 14.11. m / z(-ESI): 386.11 [M+H]-.

[0084] (6-Chloro-2,3,4-Trihydroxyphenyl)(4-Propylphenyl)methyl ketone (Compound 15)

[0085] Molecular formula: C16H15ClO4; Yellow solid, Yield: 53.7%; mp: 124.2-125.1 ℃; 1H NMR (400 MHz, CDCl3) δ 12.59 (s, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.35 (d, J =8.1 Hz, 2H), 7.30 (s, 1H), 2.74 – 2.68 (m, 2H), 1.73 (dq, J =14.7, 7.4 Hz, 2H), 1.01 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ =199.81, 150.28, 147.72, 145.85, 134.84, 132.63, 129.25, 128.62, 125.03,113.16, 111.44,38.04, 24.23, 13.80. m / z(-ESI): 303.26 [M+H]-.

[0086] (6-Bromo-2,3,4-Trihydroxyphenyl)(4-Propylphenyl)methyl ketone (Compound 14)

[0087] Molecular formula: C16H15BrO4; Yellow solid, Yield: 42.5%; mp: 72.6-73.8 ℃; 1H NMR (400 MHz, CDCl3) δ 12.59 (s, 1H), 7.62 (d, J = 8.1 Hz, 2H),7.45 (s, 1H), 7.35 (d, J = 8.1 Hz,2H), 2.75 – 2.68 (m, 2H), 1.78 – 1.68 (m,2H), 1.01 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ = 199.72, 150.70,147.75, 146.75,134.82, 132.41, 129.28, 128.63, 128.04, 114.12, 99.41, 77.34,77.03, 76.71, 38.04, 24.23, 13.81. m / z(-ESI): 352.76 [M+H]-.

[0088] 2,3-Dihydroxy-4-(4-isopropylbenzyl)phenylbutyrate (Compound 16)

[0089] Molecular formula: C20H22O5; Yellow solid, Yield: 35.7%; mp: 125.7-126.2 ℃; 1H NMR (400 MHz, CDCl3) δ 12.81 (s, 1H), 7.56 (d, J = 8.1 Hz, 2H),7.43 (d, J = 8.9 Hz, 1H), 7.33 (d, J =8.1 Hz, 2H), 6.46 (d, J = 9.0 Hz, 1H), 3.74 – 3.60 (m, 1H), 2.66 (t, J = 7.4 Hz, 2H), 1.89 – 1.79(m, 2H), 1.30 (d, J= 4.1 Hz, 3H), 1.27 (d, J = 5.1 Hz, 3H), 1.08 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ =200.18, 171.37, 157.19, 154.44, 153.25, 135.45, 132.18,129.61, 129.32, 126.47, 113.99, 107.25, 77.36, 77.04, 76.73, 35.70, 34.24,29.72, 23.76, 18.55, 13.62.m / z(-ESI): 340.41 [M+H]-.

[0090] (6-Bromo-2,3,4-Trihydroxyphenyl)(4-Isopropylphenyl)methyl ketone (Compound 17)

[0091] Molecular formula: C16H15BrO4; Yellow solid, Yield: 40.7%; mp: 130.8-131.2 ℃; 1H NMR (400 MHz, DMSO) δ 11.86 (s, 1H), 7.60 (d, J = 8.2 Hz, 2H), 7.44 (d, J =8.2 Hz, 2H), 7.15 (s, 1H), 3.06 – 2.91 (m, 1H). 13C NMR (101 MHz, DMSO) δ =198.60, 153.38, 151.33, 149.90, 135.65, 134.44, 129.62, 126.95,126.92, 114.53, 100.52, 40.63, 40.42, 40.21, 40.00, 39.79, 39.58, 39.38,33.95, 24.02.m / z(-ESI): 352.94 [M+H]-.

[0092] (2-Bromo-4,5-dihydroxyphenyl)(4-isopropylphenyl)methyl ketone (compound 18)

[0093] Molecular formula: C16H15ClO4; Yellow solid, Yield: 42.0%; mp: 123.2-124.1 ℃; 1H NMR (400 MHz, DMSO) δ 11.80 (s, 1H), 7.61 (d, J = 8.2 Hz, 2H),7.43 (d, J =8.2 Hz, 2H), 7.00 (s, 1H), 3.06 – 2.90 (m, 1H), 1.25 (d, J = 6.9Hz, 6H). 13C NMR (101 MHz, DMSO) δ = 198.62, 153.38, 150.79, 148.86, 135.66,134.71,129.63, 126.91, 123.84, 113.68, 111.94, 40.63, 40.42, 40.21, 40.00,39.79, 39.58, 39.38, 33.95, 24.01. m / z(-ESI): 307.02 [M+H]-.

[0094] Experiments verified the inhibitory effect of the target compound on hepatocyte ferroptosis:

[0095] (1) Preparation of culture media and commonly used solutions:

[0096] ① Complete culture medium: blank RPMI 1640 medium + 10% fetal bovine serum (FBS) + 1% penicillin and streptomycin (10000 U / mL each);

[0097] ② MTT solution: Weigh out MTT powder and dissolve it in PBS solution to prepare a solution with a concentration of 5.0 mg / mL. Filter to remove bacteria and store at 4°C in the dark.

[0098] ③ Preparation of compound and positive control drug: Accurately weigh an appropriate amount of compound and positive control drug fenofibrate and prepare a stock solution with a concentration of 100 μmol / L using DMSO. Before use, use blank RPMI 1640 culture medium to serially dilute the stock solution to a series of concentrations such as 25, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, and 0.005 μmol / L.

[0099] (2) Cytotoxicity test:

[0100] Take cells in the logarithmic growth phase at 1×10 4 Eighteen compounds were seeded at a density of 100 μL / well in 96-well plates and incubated at an insulated temperature for 24 h. The resulting solutions were then prepared into 25 µmol·L⁻¹ blank medium using 1640. -1 As the drug-treated group, the blank control group was added with blank 1640 medium and cultured for 24 h. The old medium was then discarded, and 100 μL / well of blank 1640 medium was added, followed by 10 μL of 5 mg / mL MTT. Cultured for another 4 h, the medium in each well was discarded, and 100 μL / well of DMSO was added and mixed. The OD value was measured at 570 nm or 490 nm using a microplate reader. Cell viability was calculated for each group as follows: Cell viability (%) = OD of drug-treated group / OD of blank group × 100%, to investigate the cytotoxic effect of the compound.

[0101] (3) The inhibitory effect of the target compound on Erastin-induced hepatocyte ferroptosis:

[0102] Erastin is a ferroptosis activator that inhibits glutathione (GSH) accumulation by suppressing cysteine ​​and glutamate transporters, leading to ferroptosis. In this invention, Erastin is used to incubate cells and induce ferroptosis in hepatocytes. First, the inhibitory effect of the target compound on hepatocyte ferroptosis is preliminarily assessed by detecting cell viability values ​​in different groups. Then, the inhibitory effect of the target compound on Erastin-induced hepatocyte ferroptosis is further assessed by detecting the levels of glutathione (GSH) and malondialdehyde (MDA), intracellular reactive oxygen species (ROS), iron ion content, and mitochondrial number in the cell culture supernatant. The extent of hepatocyte damage is assessed by detecting the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in the cell culture supernatant. Finally, cell morphology is observed to more visually demonstrate the inhibitory effect of the target compound on ferroptosis.

[0103] Cells in logarithmic growth phase were fed at a rate of 1×10⁻⁶. 4 Eighteen compounds were seeded at a density of 100 μL / well in 96-well cell culture plates and cultured for 24 h. Then, 18 compounds were prepared using 1640 blank culture medium at concentrations of 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, and 0.005 µmol·L⁻¹. -1 The solution contained fenofibrate as a positive control. Experimental groups: ① Blank control group: serum-free 1640 culture medium; ② Model group: 30 µmol·L⁻¹ -1 Erastin, serum-free 1640 culture medium; ③ Drug treatment group: 30 µmol·L -1 Erastin, different concentrations of the test compound prepared above; ④ Positive control group: 30 µmol·L -1 Erastin, the positive control drug fenofibrate concentration is 12 µmol·L⁻¹ -1 All groups were incubated at 37℃ for 4 h; afterwards, the model group, drug-treated group, and positive control group were incubated at 30 µmol·L⁻¹. -1 Erastin was incubated at 37°C for another 4 hours. Cell viability was assessed using the MTT assay, and the protective effect of the compound against hepatocyte injury was calculated. EC50 was calculated using Graphpad Prism 8. 50 Value. Protection rate (%) = (OD treatment group - OD model group) / (OD control group - OD model group) × 100%.

[0104] (3) Determination of ALT, AST, LDH, GSH, and MDA levels in cell culture supernatant, as well as determination of intracellular ROS, iron ion content, and mitochondrial number:

[0105] 1×10 5HL-02 cells at 1 / mL in the logarithmic growth phase were seeded at 200 μL / well into 6-well plates. After 24 h of culture, the experimental groups were: ① Blank control group: serum-free 1640 medium; ② Model group: 30 µmol·L⁻¹ medium. -1 Erastin, serum-free 1640 culture medium; ③ Drug treatment group: 30 µmol·L -1 Erastin, with its hepatocellular injury protection rate and EC50. 50 The three representative compounds, compound 5, compound 13, and compound 18, with relatively good values, were prepared in a 10 µmol·L⁻¹ solution. -1 ④ Positive control group: 30 µmol·L -1 Erastin, the positive control drug fenofibrate concentration is 12 µmol·L⁻¹ -1 All groups were incubated at 37℃ for 4 hours; afterwards, the model group, drug-treated group, and positive control group were incubated at 30 µmol·L⁻¹. -1 Erastin was used to culture the cells at 37°C for another 4 hours. The cell supernatant was collected from the 6-well plates, and the contents of ALT, AST, LDH, GSH, and MDA, as well as the contents of ROS, iron ions, and mitochondria in the cell supernatant were measured according to the kit instructions.

[0106] (4) Cell morphology observation:

[0107] Cells in logarithmic growth phase were fed at a rate of 1×10⁻⁶. 5 Fenofibrate was seeded at a density of 200 μL / well in 6-well plates at a density of 1 / mL and cultured for 24 h. After incubation, fenofibrate was a positive result. Experimental groups: ① Blank control group: serum-free 1640 medium; ② Model group: 30 µmol·L⁻¹ medium. -1 Erastin, serum-free 1640 culture medium; ③ Drug treatment group: 30 µmol·L -1 Erastin prepared a 10 µmol·L⁻¹ concentration using the stock solutions of compounds 5, 13, and 18 with 1640 blank culture medium. -1 ④ Positive control group: 30 µmol·L -1 Erastin, the positive control drug fenofibrate concentration is 12 µmol·L⁻¹ -1 All groups were incubated at 37℃ for 4 hours; afterwards, the model group, drug-treated group, and positive control group were incubated at 30 µmol·L⁻¹. -1 The cells were cultured in Erastin at 37°C for another 4 hours, and then the cells were removed and observed under an inverted microscope to observe their morphology.

[0108] (5) Statistical analysis:

[0109] All experiments were repeated three times in parallel. Results are expressed as mean ± standard deviation (M ± SD) of the three parallel trials. T-tests were used to analyze the statistical data. p <0.05 indicates a significant difference. p <0.01 indicates a highly significant difference.

[0110] (6) Toxicity of the target compound:

[0111] The cytotoxicity results of 18 compounds are as follows Figure 5 As shown, the results indicate that 18 compounds were effective at 25 µmol·L⁻¹. -1 After treatment of hepatocytes at the concentration of HL-02 for 24 hours, there was no significant difference in cell viability compared with the blank control group. p >0.05), indicating that 18 compounds were at 25 µmol·L⁻¹. -1 At concentrations of [specific concentration], the compound had no effect on the survival of HL-02 hepatocytes, suggesting that it was essentially non-cytotoxic at these concentrations.

[0112] (7) The target compound inhibits ferroptosis in HL-02 hepatocytes:

[0113] ① The target compound enhances cell viability:

[0114] The results of the target compounds inhibiting Erastin-induced hepatocyte ferroptosis are shown in Table 1. The results showed that all 18 compounds had varying degrees of protective effect against hepatocytes. In terms of cell protection rate, 17 compounds had a higher protection rate than the positive control drug fenofibrate (47.67% ± 0.0085), with 4 of these compounds having a protection rate higher than 99.9%. Among these 17 compounds with higher protection rates than the positive control drug, 13 compounds had an EC50 value of [missing information]. 50 The value was lower than that of the lead compound SD (3.8486 ± 0.0664 µmol·L⁻¹). -1 EC of 11 compounds 50 The value was lower than that of the positive control drug fenofibrate (1.634±0.0311µmol·L⁻¹). -1 It is worth noting that the EC50 of the three compounds 50 The values ​​reach the nanomolar order: they are 9.1 ± 0.0022 nmol·L⁻¹. -1 6.8 ± 0.0091 nmol·L -1 5.8±0.0013 nmol·L -1 Taking into account the nanomolar order of magnitude of EC 50 Based on the two indicators of high protective activity (over 99.9%), three representative compounds, compound 5, compound 13 and compound 18, were selected for further in-depth study in subsequent experiments.

[0115] Table 1. Inhibitory effect of target compounds on hepatocyte ferroptosis

[0116] Compd. <![CDATA[Protective Rate(%) # ]]> <![CDATA[EC 50 (µmol·L -1 ) # ]]> Compound 1 >99.9% 0.9554±0.0673 Compound 2 61.15%±0.0018 0.6989±0.0258 Compound 3 42.33%±0.0011 10.6733±0.0709 Compound 4 60.56%±0.0054 0.1444±0.0040 Compound 5 >99.9% 0.7957±0.0113 Compound 6 77.49%±0.0014 0.3878±0.0391 Compound 7 63.27%±0.0171 0.0091±0.0022 Compound 8 93.87±0.0165 0.3256±0.1020 Compound 9 >99.9% 4.2853±0.0072 Compound 10 69.02%±0.0056 0.0796±0.0148 Compound 11 56.58%±0.0016 31.07±0.0458 Compound 12 91.99%±0.0016 1.8493±0.0066 Compound 13 61.58%±0.0119 0.0068±0.0091 Compound 14 81.41%±0.0010 20.46±0.1609 Compound 15 76.74%±0.0014 14.86±0.1039 Compound 16 95.1%±0.0021 1.311±0.0888 Compound 17 61.43%±0.0037 0.2289±0.0347 Compound 18 >99.9% 0.0058±0.0013 Fenofibrate 47.67%±0.0085 1.634±0.0311 SD >99.9% 3.8486±0.0664

[0117] ②The target compound alleviates hepatocellular damage by inhibiting Erastin-induced ferroptosis:

[0118] Iron ions are a key factor in triggering ferroptosis. Abnormal accumulation of iron ions in cells promotes the production of lipid peroxidation products and reduces the number of mitochondria. As expected, treatment of HL-02 cells with the ferroptosis inducer Erastin increased intracellular iron ion levels as well as the levels of ROS, MDA, ALT, AST, and LDH. Figure 6-11 As shown, the number of mitochondria and the content of GSH are significantly reduced, such as Figure 12-13 As shown, HL-02 cells underwent ferroptosis, resulting in severe cell damage. Treatment with the target compound reversed this trend. Compared to the model group, the treated cells exhibited significantly lower iron ion, ROS, and MDA levels, and significantly increased mitochondrial number and GSH levels, indicating that the target compound inhibited ferroptosis. Furthermore, the treated cells showed significantly reduced levels of ALT, AST, and LDH, suggesting that the target compound can alleviate hepatocyte damage by inhibiting ferroptosis.

[0119] ③ Effects of the target compound on hepatocyte morphology:

[0120] like Figure 14 As shown, HL-02 cells in the normal group exhibited normal morphology and good adherence and growth. After Erastin treatment, the cells became rounded and shrunken, with a significant increase in floating cells and a marked increase in the number of dead cells. After treatment with the target compound, the cell morphology recovered, adherence was good, and growth was somewhat restored. This further demonstrates that the target compound can inhibit ferroptosis. Figure 14 In the diagram, a represents the normal control group, b represents the model group, c represents the Bifendate group, d represents the compound 13 administration group, e represents the compound 18 administration group, and f represents the compound 5 administration group. The dosage for d, e, and f is 10 µmol·L⁻¹. -1 .

[0121] This invention synthesizes 18 polyhydroxy-substituted benzophenone derivatives, none of which have been reported in the literature. Due to their unique structure, they can inhibit ferroptosis and thus exert excellent hepatoprotective effects. After incubating cells, the polyhydroxy-substituted benzophenone derivatives contained in this invention can reduce the accumulation of intracellular iron ions and reactive oxygen species, restore mitochondrial activity, alleviate cell damage, and effectively inhibit hepatocellular ferroptosis induced by the ferroptosis inducer Erastin.

Claims

1. A polyhydroxy-substituted benzophenone derivative, characterized in that: The structural formula is: In this context, R1 = ethyl, n-propyl, isopropyl; R2 = hydrogen, bromine, chlorine; and R3 = butyryloxy, morpholine-4-carbonyloxy, hydroxyl.

2. The polyhydroxy-substituted benzophenone derivative according to claim 1, characterized in that: The structural formula is: In this context, R1 = ethyl, n-propyl, isopropyl, R2 = hydrogen, bromine, chlorine, and R3 = hydroxyl.

3. The polyhydroxy-substituted benzophenone derivative according to claim 1, characterized in that: The structural formula is: In this context, R1 = ethyl, n-propyl, isopropyl, R2 = hydrogen, and R3 = butyryloxy, morpholine-4-carbonyloxy, and hydroxyl.

4. Application of polyhydroxy-substituted benzophenone derivatives in preventing hepatocyte ferroptosis. The structural formula of polyhydroxy-substituted benzophenone derivatives is as follows: ,in, R1 = ethyl, n-propyl, isopropyl; R2 = hydrogen, bromine, chlorine; R3 = butyryloxy, morpholine-4-carbonyloxy, hydroxyl.

5. The application of the polyhydroxy-substituted benzophenone derivatives according to claim 4 in preventing hepatocyte ferroptosis, characterized in that: The structural formulas of polyhydroxy-substituted benzophenone derivatives are as follows: In this context, R1 = ethyl, n-propyl, isopropyl, R2 = hydrogen, bromine, chlorine, and R3 = hydroxyl.

6. The application of the polyhydroxy-substituted benzophenone derivatives according to claim 5 in preventing hepatocyte ferroptosis, characterized in that: The structural formulas of polyhydroxy-substituted benzophenone derivatives are as follows: .

7. The application of the polyhydroxy-substituted benzophenone derivatives according to claim 4 in preventing hepatocyte ferroptosis, characterized in that: The structural formulas of polyhydroxy-substituted benzophenone derivatives are as follows: In this context, R1 = ethyl, n-propyl, isopropyl, R2 = hydrogen, and R3 = butyryloxy, morpholine-4-carbonyloxy, and hydroxyl.

8. The application of the polyhydroxy-substituted benzophenone derivatives according to claim 7 in preventing hepatocyte ferroptosis, characterized in that: The structural formulas of polyhydroxy-substituted benzophenone derivatives are as follows: .

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