EGC-based glutamate dehydrogenase degradation agent

Abstract

The invention belongs to the technical field of drug synthesis, and provides an EGC-based glutamate dehydrogenase degradation agent which comprises a structural general formula I, II or III. A series of conjugates of EGC and a protein degradation targeting chimera are synthesized to achieve the effect of intracellular GDH protein degradation, and GDH protein degradation can prevent degradation of a large amount of glutamic acid, so that toxicity to nerve cells caused by generation of a large amount of ammonia is prevented; and a new strategy is provided for treatment of rare diseases of the high insulin-hyperamminemia syndrome and treatment of other diseases related to high expression of GDH. In order to improve the biological activity of a GDH inhibitor, the invention provides a series of degradation agents based on a PROTAC technology, namely EGCG and EGC derivatives, the degradation agents comprise protein degradation targeting chimera molecules, and GDH protein is more effectively degraded by utilizing an intracellular ubiquitin proteasome system.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical synthesis technology, and more particularly to a glutamate dehydrogenase degrading agent based on EGC. Background Technology

[0002] Alterations or dysregulation of glutamate dehydrogenase (GDH) function can lead to diseases such as hyperinsulinemia-hyperammonia, Alzheimer's disease, and cancer. For example, hyperinsulinemia-hyperammonia syndrome (HHS) is a rare disease characterized by fasting and protein-sensitive hypoglycemia in children, accompanied by persistent hyperammonemia. Enhanced mutations in GDH function are believed to cause HHS, and high GDH activity is also observed in neurological diseases such as Alzheimer's disease (AD). However, current treatments for HHS are mostly symptomatic, and these drugs are ineffective in controlling hyperammonemia and central nervous system lesions. Therefore, a more ideal option is to find a GDH inhibitor for direct treatment of this disease. Currently, no GDH inhibitor has entered clinical trials.

[0003] Currently reported GDH inhibitors include EGCG (partial inhibitors 50%) and selenium compounds (IC). 50 0.5 μM), small molecule compound GW5074 (IC 50 The inhibitors are 1.5 μM, HCP (1.7 μM), BTH (5.5 μM), and R162 (>10 μM). These only inhibit GDH enzymes but cannot degrade GDH. Therefore, designing and synthesizing a series of degradative agents based on PROTAC technology based on EGC is of great significance in improving the bioactivity of GDH inhibitors. Summary of the Invention

[0004] The purpose of this invention is to provide an EGC-based glutamate dehydrogenase degrading agent in order to overcome the shortcomings of the prior art.

[0005] The beneficial effects of this invention include:

[0006] This invention synthesizes a series of EGC-PROTAC conjugates to enhance their ability to degrade GDH protein, thereby achieving intracellular GDH protein degradation. This prevents the degradation of large amounts of glutamate and the toxic effects of excessive ammonia production on nerve cells. These conjugates are intended for the treatment of the rare disease hyperinsulinemia-hyperammonia syndrome (HHS) and other diseases associated with high GDH expression, providing a new strategy for the treatment of HHS. To improve the bioactivity of GDH inhibitors, this invention provides a series of PROTAC-based degradative agents—EGCG and EGC derivatives—containing protein degradation-targeting chimeric molecules. These molecules utilize the intracellular ubiquitin-proteasome system to more effectively degrade GDH protein. Compounds EGC-2, EGC-3, EGC-12, and EGC-15 possess the function of degrading both GDH and GAPDH proteins and participate in cellular energy metabolism, apoptosis, and anti-tumor activities. Attached Figure Description

[0007] Figure 1 The graph shows the inhibitory activity curves of different concentrations and types of degrading agents on glutamate dehydrogenase.

[0008] Figure 2 The results show the degradation of GDH and GAPDH at the cellular level by an EGC-based glutamate dehydrogenase degrader.

[0009] Figure 3 The graph shows the inhibitory activity of EGC-based glutamate dehydrogenase degraders on H22 tumor cells.

[0010] Figure 4 The effect of different concentrations of glutamate dehydrogenase degrading agents on the survival rate of PC12 cells under different concentrations of glutamate treatment. Detailed Implementation

[0011] This invention provides an EGC-based glutamate dehydrogenase degrading agent, which is formed by linking EGC-like compounds with PROTAC-like small molecules. The degrading agent comprises the following general structural formula:

[0012]

[0013] Where X is independent, it is O or NH; Y and Z are independent, they are F or H; and L is... n1 is 1, 2, 3 or 4, n2 is 2, 3, 4 or 5, n3 is 5, 7 or 9, and n4 is 5, 7 or 9.

[0014] In this invention, the compound of general structural formula I preferably comprises the following structural formula:

[0015]

[0016] In this invention, compounds of general structural formula II preferably include the following structural formulas:

[0017]

[0018]

[0019]

[0020] In this invention, compounds of general formula III preferably include the following structural formulas:

[0021]

[0022]

[0023]

[0024] The present invention also provides the application of the aforementioned EGC-based glutamate dehydrogenase degrading agent in the preparation of drugs that degrade GDH protein.

[0025] In this invention, EGC-2, EGC-3, EGC-12, and EGC-15 are used to prepare drugs that degrade the GDH and GAPDH dual proteins.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1: Synthesis of EGC-1

[0028]

[0029] Reagents and reaction conditions: a: diethylene glycol bis(p-toluenesulfonate), NaH, DMF; b: 4-hydroxythalidomide, NaHCO3, NaI, DMF; c: H2, Pd / C, tetrahydrofuran / methanol.

[0030] Synthesis of Intermediate 2: 4.4 mmol of epigallocatechin gallate was dissolved in 30 mL of N,N-dimethylformamide, followed by the addition of 87 mmol of potassium carbonate and 6.2 mL of benzyl bromide. The mixture was stirred at room temperature for 48 h. TLC (petroleum ether:ethyl acetate = 5:1) confirmed the reaction was complete. The reaction solution was poured into water, and the pH was adjusted to neutral with dilute hydrochloric acid. The solution was extracted three times with 30 mL of ethyl acetate. The ethyl acetate extract was washed four times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Recrystallization was performed with 80 mL of diethyl ether, and the resulting solution was filtered to give a white solid, with a yield of 37.7%.

[0031] Synthesis of Intermediate 3: 0.42 mmol of Intermediate 2 was dissolved in a mixture of 5 mL methanol and 5 mL ethylene glycol dimethyl ether. 1.4 mmol of potassium carbonate was added, and the mixture was stirred at room temperature for 24 h. TLC (petroleum ether:ethyl acetate = 5:1) confirmed the reaction was complete. Silica gel was added, and the mixture was subjected to column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain a white solid with a yield of 60%. If the product after column chromatography contains a small amount of impurities, it can be slurried using a 10:1 volume ratio of petroleum ether and ethyl acetate, filtered, and dried.

[0032] Synthesis of EGC-1: 1 mmol of intermediate 3 was added to a 50 mL three-necked flask, dissolved in 20 mL of N,N-dimethylformamide, and then 3 mmol of sodium hydride (60%) was added to replace N2. The mixture was stirred at 0 °C for 30 min, and the solution gradually turned yellow. 4 mmol of diethylene glycol bis(p-toluenesulfonate) was dissolved in 10 mL of N,N-dimethylformamide and added to the system of compound 3 under a nitrogen atmosphere. The mixture was naturally heated to room temperature and stirred overnight. After the reaction was complete as monitored by TLC, excess sodium hydride was quenched by adding 0.1 N dilute hydrochloric acid. The mixture was extracted three times with ethyl acetate, and the ethyl acetate extracts were combined and washed four times with saturated brine. The ethyl acetate extract was dried over anhydrous sodium sulfate and filtered. Silica gel was added and the mixture was stirred by column chromatography to obtain intermediate 4 in 67% yield.

[0033] 0.93 mmol of intermediate 4 and 2.79 mmol of 4-hydroxythalidomide were dissolved in 15 mL of N,N-dimethylformamide, followed by the addition of 1.86 mmol of sodium bicarbonate and 0.93 mmol of sodium iodide. The mixture was stirred overnight at 80 °C. The reaction was monitored by TLC until completion. The reaction mixture was poured into water and extracted three times with ethyl acetate, followed by washing the ethyl acetate extract four times with saturated brine. The ethyl acetate extract was dried over anhydrous sodium sulfate and filtered. Silica gel was added and the mixture was subjected to column chromatography to obtain a yellow oil. The yellow oil was dissolved in a tetrahydrofuran / methanol mixture, and palladium on carbon was added. The air was replaced with hydrogen, and the mixture was stirred overnight at room temperature. After filtration, the solvent was removed by rotary evaporation using a conventional water pump to obtain a pale yellow solid EGC-1, with a yield of 96%.

[0034] 1H NMR(400MHz,DMSO-d6)δ11.10(s,1H),9.17(s,1H),8.95(s,1H),8.75(s,2H),8.01(s,1H),7.78( t,J=8.0Hz,1H),7.44(dd,J=8.8,4.8Hz,2H),6.41(s,2H),5.89(d,J=2.0Hz,1H),5.72(d,J=2.0Hz ,1H),5.07(dd,J=12.8,5.6Hz,1H),4.79(s,1H),4.24–4.18(m,2H),3.93–3.83(m,1H),3.69–3.57 (m,3H),3.54–3.43(m,3H),3.34(s,1H),2.95–2.80(m,1H),2.65–2.52(m,3H),2.06–1.96(m,1H).

[0035] 13 C NMR(101MHz,DMSO)δ173.31,170.47,167.31,165.75,156.94,156.80,156.29,156.11,145.92(2C),137.48,133.66,132.57,129.8 8,120.39,116.70,115.78,106.27(2C),98.70,95.56,94.50,77.34,74.49,70.53,69.29,69.10(2C),49.18,31.42,25.60,24.92.

[0036] HRMS(ESI): m / z calcd for C 32 H 34 N3O 13 + 668.2086[M+NH4] + ;found:668.2089.

[0037] Example 2: Synthesis of EGC-2

[0038] In the synthesis of EGC-1, diethylene glycol bis-toluenesulfonate was replaced with triethylene glycol bis-toluenesulfonate, while other conditions remained the same as the synthesis process of EGC-1.

[0039] 1H NMR (400MHz, DMSO-d6) δ11.10(s,1H),9.19(s,1H),8.96(s,1H),8.73(s,2H),8.00(s,1H),7.78(t,J=8.0H z,1H),7.49(d,J=8.4Hz,1H),7.44(d,J=7.2Hz,1H),6.39(s,2H),5.89(d,J=2.0Hz,1H),5.72(d,J=2.4Hz,

[0040] 1H),5.08(dd,J=12.8,5.6Hz,1H),4.78(s,1H),4.30(t,J=4.4Hz,2H),3.85(t,J=4.4Hz,1H),3.74(t,J=4 .4Hz,2H),3.64–3.43(m,5H),3.37–3.27(m,4H),2.94–2.81(m,1H),2.64–2.55(m,3H),2.06–1.98(m,1H).

[0041] 13 C NMR (101MHz, DMSO) δ173.31,170.46,167.30,165.76,156.92,

[0042] 156.80,156.31,156.08,145.91(2C),137.47,133.68,132.56,129.81,120.48,116.72,115.82,106.25(2C) ,98.72,95.56,94.50,77.30,74.54,70.46,70.28,70.24,69.25,69.18,69.08,49.19,31.42,25.60,25.05.

[0043] HRMS(ESI): m / z calcd for C 34 H 38 N3O 14 + 712.2348[M+NH4] + ;found:712.2353.

[0044] Example 3: Synthesis of EGC-3

[0045] In the synthesis of EGC-1, diethylene glycol bis-toluenesulfonate was replaced with tetraethylene glycol bis-toluenesulfonate, while other conditions remained the same as the synthesis process of EGC-1.

[0046] 1H NMR (400MHz, DMSO-d6) δ11.10(s,1H),9.17(s,1H),8.93(s,1H),8.71(s,2H),7.98(s,1H),7.79(dd,J=8.4,7.2Hz, 1H),7.50(d,J=8.6Hz,1H),7.45(d,J=7.2Hz,1H),6.38(s,2H),5.89(d,J=2.4Hz,1H),5.71(d,J=2.4Hz,1H),5.08( dd,J=12.8,5.2Hz,1H),4.78(s,1H),4.37–4.28(m,2H),3.86–3.81(m,1H),3.80–3.76(m,2H),3.63–3.57(m,3H),3 .51–3.36(m,8H),3.33–3.27(m,2H),2.89(s,1H),2.61(t,J=4.4Hz,2H),2.57(d,J=4.8Hz,1H),2.06–1.99(m,1H).

[0047] 13 C NMR(101MHz,DMSO)δ173.30,170.44,167.30,165.76,156.92,156.80,156.30,156.08,145.91(2C),137.47,133.69,132.57,129.80,120.48,1 16.74,115.85,106.24(2C),98.72,95.56,94.50,77.30,74.56,70.56, 70.25,70.23,70.14,69.26,69.11,67.49,49.20,31.42,25.60,25.08.

[0048] HRMS(ESI): m / z calcd for C 36 H 42 N3O 15 + 756.2610[M+NH4] + ;found:756.2614.

[0049] Example 4: Synthesis of EGC-4

[0050] In the synthesis of EGC-1, diethylene glycol bis-toluenesulfonate was replaced with pentaethylene glycol bis-toluenesulfonate, while other conditions remained the same as the synthesis process of EGC-1.

[0051] 1H NMR (400MHz, DMSO-d6) δ11.10(s,1H),9.17(s,1H),8.94(s,1H),8.71(s,2H),7.98(s,1H),7.77(dd,J=8.4,7.2Hz, 1H),7.50(d,J=8.6Hz,1H),7.45(d,J=7.2Hz,1H),6.38(s,2H),5.89(d,J=2.4Hz,1H),5.71(d,J=2.4Hz,1H),5.09( dd,J=12.8,5.2Hz,1H),4.78(s,1H),4.37–4.28(m,2H),3.86–3.81(m,1H),3.80–3.76(m,2H),3.63–3.57(m,3H),3 .51–3.36(m,10H),3.33–3.27(m,4H),2.89(s,1H),2.61(t,J=4.4Hz,2H),2.57(d,J=4.8Hz,1H),2.06–1.99(m,1H).

[0052] HRMS(ESI): m / z calcd for C 38 H 36 N3O 14 + 800.2572[M+NH4] + ;found:712.2602.

[0053] Example 5: Synthesis of EGC-5

[0054]

[0055] Reagents and reaction conditions: a: 3-bromopropyne, NaH, tetrahydrofuran; b: CuI, N-ethyldiisopropylamine, 6-azidohexanoic acid pomadiolamine; c: H2, Pd / C, tetrahydrofuran / methanol.

[0056] 0.4 mmol of intermediate 3 was dissolved in tetrahydrofuran and stirred at 0 °C for 10 min. 1.5 mmol of 60% sodium hydride was added under a nitrogen atmosphere and stirred for 30 min. 0.48 mmol of 3-bromopropyne was added to the reaction system, and the mixture was slowly heated to room temperature and stirred for 24 h. After the reaction was complete as monitored by TLC, the pH was adjusted to neutral by adding 1 N dilute hydrochloric acid. The mixture was extracted with ethyl acetate, washed four times with water, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by column chromatography to give a white solid, intermediate 5, in 85% yield.

[0057] 0.36 mmol of intermediate 5 was dissolved in 3 mL of hexamethylphosphoric acid triamine. 0.30 mmol of pomalidoamine 6-azidohexanoate, 2 mL of N-ethyldiisopropylamine, and 7.8 mg of cuprous iodide were added to the system, and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC until complete. 2N dilute hydrochloric acid was added to the system, resulting in the precipitation of a white solid. The filter cake was collected by filtration and dried. The filter cake was dissolved in a dichloromethane / methanol mixture, mixed with silica gel, and subjected to column chromatography to obtain a white solid—intermediate 6—in 60% yield.

[0058] Intermediate 6 was dissolved in a tetrahydrofuran / methanol mixture, and palladium on carbon was added to displace hydrogen. The mixture was stirred overnight at room temperature. After filtration, the solvent was removed by vacuum distillation to give a pale yellow solid EGC-5 in 98% yield.

[0059] 1 H NMR(400MHz,DMSO-d6)δ11.10(s,1H),9.17(s,1H),9.07(s,1H),8.93(s,1H),8.71(s,2H),7.98(s,1H),7.79(d d,J=8.4,7.2Hz,1H),7.50(d,J=8.6Hz,1H),7.45(d,J=7.2Hz,1H),6.38(s,2H),5.89(d,J=2.4Hz,1H),5.71(d, J=2.4Hz,1H),5.08(dd,J=12.8,5.2Hz,1H),5.06(s,2H),4.78(s,1H),4.36(t,J=7.2Hz,2H),2.89(s,1H),2.61 (t,J=4.4Hz,2H),2.57(d,J=4.8Hz,1H),2.06–1.99(m,1H),1.89–1.79(m,2H),1.70–1.59(m,2H),1.28(m,2H).

[0060] HRMS(ESI): m / z calcd for C 37 H 40 N7O 12 + 774.2729[M+NH4] + ;found:774.2731.

[0061] Example 6: Synthesis of EGC-6

[0062] In the synthesis of EGC-5, 6-azidohexanoate pomadoamine was replaced with 8-azidooctanoate pomadoamine, and the other conditions were the same as those in the synthesis process of EGC-5. The yield of the second step was 48%.

[0063] 1H NMR(400MHz,DMSO-d6)δ11.10(s,1H),9.16(s,1H),9.07(s,1H),8.83(s,1H),8.71(s,2H),7.98(s,1H),7.79(dd ,J=8.4,7.2Hz,1H),7.50(d,J=8.6Hz,1H),7.55(d,J=7.2Hz,1H),6.38(s,2H),5.89(d,J=2.4Hz,1H),5.71(d,J= 2.4Hz,1H),5.18(dd,J=12.8,5.2Hz,1H),5.06(s,2H),4.78(s,1H),4.36(t,J=7.2Hz,2H),2.89(s,1H),2.63(t, J=4.4Hz,2H),2.57(d,J=4.8Hz,1H),2.06–1.99(m,1H),1.89–1.79(m,2H),1.70–1.59(m,2H),1.28-1.20(m,6H).

[0064] HRMS(ESI): m / z calcd for C 39 H 44 N7O 12 + :802.3042[M+NH4] + ;found:802.3045.

[0065] Example 7: Synthesis of EGC-7

[0066] In the synthesis of EGC-5, 6-azidohexanoic acid pomadoamine was replaced with 10-azidodecanoic acid pomadoamine, and the other conditions were the same as those for the synthesis of EGC-5, with a yield of 40%.

[0067] 1H NMR(400MHz,DMSO-d6)δ11.11(s,1H),9.18(s,1H),9.07(s,1H),8.93(s,1H),8.71(s,2H),7.98(s,1H),7.79(dd ,J=8.4,7.2Hz,1H),7.52(d,J=8.6Hz,1H),7.46(d,J=7.2Hz,1H),6.39(s,2H),5.89(d,J=2.4Hz,1H),5.71(d,J=2 .4Hz,1H),5.08(dd,J=12.8,5.2Hz,1H),5.06(s,2H),4.78(s,1H),4.36(t,J=7.2Hz,2H),2.89(s,1H),2.62(t,J =4.4Hz,2H),2.57(d,J=4.8Hz,1H),2.06–1.99(m,1H),1.89–1.79(m,2H),1.70–1.59(m,2H),1.28-1.20(m,10H).

[0068] HRMS(ESI): m / z calcd for C 41 H 48 N7O 12 + 830.3355[M+NH4] + Found: 830.3359.

[0069] Example 8: Synthesis of EGC-15

[0070]

[0071] Reagents and reaction conditions: a) CuI, K2CO3, DMF; b) H2, Pd / C, THF / MeOH.

[0072] 1H NMR (400MHz, DMSO-d6) δ11.10(s,1H),9.17(s,1H),8.94(s,1H),8.71(s,2H),7.99(s,1H),7.79(dd,J=8. 4,7.2Hz,1H),7.52(d,J=8.6Hz,1H),7.46(d,J=7.2Hz,1H),6.38(s,2H),5.89(d,J=2.4Hz,1H),5.71(d,J= 2.4Hz,1H),5.08(dd,J=12.8,5.2Hz,1H),4.78(s,1H),4.36(t,J=7.2Hz,2H),3.98–3.86(m,2H),2.89(s,1 H),2.61(t,J=4.4Hz,2H),2.57(d,J=4.8Hz,1H),2.06–1.99(m,1H),1.71–1.62(m,4H),1.49–1.41(m,2H).

[0073] HRMS(ESI): m / z calcd for C 34 H 37 N4O 12 + 693.2402[M+NH4] + ;found:693.2405.

[0074] Example 9: Synthesis of EGC-16

[0075] In the synthesis of EGC-15, 6-bromohexanoic acid pomadiolamine was replaced with 8-bromooctanoic acid pomadiolamine, and other conditions were the same as those for the synthesis of EGC-15.

[0076] 1H NMR (400MHz, DMSO-d6) δ11.10(s,1H),9.16(s,1H),8.93(s,1H),8.71(s,2H),7.98(s,1H),7.79(dd,J=8. 4,7.2Hz,1H),7.51(d,J=8.6Hz,1H),7.45(d,J=7.2Hz,1H),6.39(s,2H),5.89(d,J=2.4Hz,1H),5.71(d,J= 2.4Hz,1H),5.09(dd,J=12.8,5.2Hz,1H),4.78(s,1H),4.36(t,J=7.2Hz,2H),3.98–3.86(m,2H),2.89(s,1 H),2.61(t,J=4.4Hz,2H),2.57(d,J=4.8Hz,1H),2.06–1.99(m,1H),1.70–1.62(m,4H),1.39–1.31(m,6H).

[0077] Example 10 Synthesis of EGC-11 and EGC-211

[0078]

[0079] Reagents and reaction conditions: a: EDCI, DMAP, DMF; b: NaBH4, Pd(PPh3)4, THF; c: KI, K2CO3, DMF; d: H2, Pd / C, THF / MeOH; e: 0.15% p-benzenesulfonic acid, chloroform.

[0080] 0.074 mmol of intermediate 3, 0.074 mmol of intermediate 9, 0.089 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 0.111 mmol of 4-dimethylaminopyridine (DMAP) were dissolved in 1 mL of N,N-dimethylformamide and stirred at room temperature for 48 h. The reaction was monitored by TLC until complete. The reaction mixture was poured into water and extracted three times with ethyl acetate. The ethyl acetate extract was washed four times with saturated brine, dried over anhydrous sodium sulfate, and filtered. Silica gel was added and the mixture was stirred, and column chromatography was used to give a white solid—intermediate 10—in 72% yield.

[0081] 0.336 mmol of intermediate 10 was dissolved in 10 mL of anhydrous tetrahydrofuran, and 0.067 mmol of tetra(triphenylphosphine)palladium (Pd(PPh3)4) was added. 2.01 mmol of sodium borohydride was added at 0 °C, and the mixture was slowly heated to room temperature with stirring overnight. The reaction was monitored by TLC until complete. Excess sodium borohydride was quenched with saturated ammonium chloride solution in an ice bath, and the mixture was extracted with 10 mL of ethyl acetate. The ethyl acetate extract was dried over anhydrous sodium sulfate and filtered. The sample was stirred with silica gel and subjected to column chromatography to obtain a pale yellow solid – intermediate 11 – in 49% yield.

[0082] 0.5 mmol of intermediate 11 and 0.5 mmol of pomadiolamine 6-bromohexanoate were dissolved in 5 mL of N,N-dimethylformamide. Then, 0.05 mmol of potassium iodide and 1.25 mmol of potassium carbonate were added sequentially. The mixture was stirred overnight at 80 °C, and the reaction was monitored by TLC until completion. The system was poured into water, and the aqueous phase was extracted three times with ethyl acetate. The ethyl acetate extract was washed four times with saturated brine. The ethyl acetate extract was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Silica gel was added, and the mixture was subjected to column chromatography to obtain a pale yellow oily substance—intermediate 12—in 26.2% yield.

[0083] Intermediate 12 was dissolved in a tetrahydrofuran / methanol mixture, and palladium on carbon was added to displace hydrogen. The mixture was stirred overnight at room temperature. After filtration and solvent removal by vacuum distillation, a grayish-white solid EGC-11 was obtained, with a yield of 89%.

[0084] HRMS(ESI): m / z calcdforC 44 H 40 F2N3O 17 + 920.2321[M+H] + ;found:920.23212.

[0085] EGC-11 was dissolved in chloroform, and 0.15% p-benzenesulfonic acid was added. The mixture was stirred at room temperature. After filtration, the solvent was removed by vacuum distillation to obtain a grayish-white solid, EGC-211, in 89% yield.

[0086] 1HNMR(400MHz,DMSO-d6)δ11.16(s,1H),9.70(s,1H),9.39–9.29(m,3H),9.06(s,1H),8.71(s,2H), 8.05(s,1H),7.82(t,J=8.0Hz,1H),6.82(s,2H),6.40(s,2H),5.93(d,J=2.4Hz,1H),5.82(d,J=2.4 Hz,1H),5.38(s,1H),5.14(dd,J=12.8,5.2Hz,1H),4.96(s,1H),3.98–3.86(m,2H),2.99–2.82(m,2 H),2.71–2.54(m,3H),2.48–2.43(m,2H),2.10–2.02(m,1H),1.71–1.62(m,4H),1.49–1.41(m,2H).

[0087] HRMS(ESI): m / z calcdforC 41 H 39 F2N4O 16 + 881.2323[M+NH4] + ;found:881.23232.

[0088] Example 11 Synthesis of EGC-12 and EGC-212

[0089] The synthesis of EGC-12 involved replacing 6-bromohexanoic acid pomadocromine in the synthesis of EGC-11 with 8-bromooctanoic acid pomadocromine, while other conditions remained the same as for EGC-11. The synthesis of compound EGC-212 followed the same procedure as the hydrolysis of EGC-11 to EGC-211.

[0090] EGC-12, HRMS(ESI):m / z calcd forC 46 H 44 F2N3O 17 + 948.2634 [M+H] + ;found:948.2635.

[0091] EGC-212, 1H NMR (400MHz, DMSO-d6) δ11.17(s,1H),9.70(s,1H),9.32(d,J=5.6Hz,3H),8.75(s,2H),8.47(d,J=8.4Hz,1H ),7.83(t,J=8.0Hz,1H),7.61(d,J=7.2Hz,1H),6.83(s,2H),6.42(s,2H),5.94(d,J=2.4Hz,1H),5.84(d,J= 2.0Hz,1H),5.44–5.34(m,1H),5.16(dd,J=12.8,5.6Hz,1H),4.98(s,1H),3.90(t,J=6.8Hz,2H),3.01–2.83 (m,2H),2.73–2.53(m,3H),2.46(t,J=7.6Hz,2H),2.12–2.02(m,1H),1.70–1.56(m,4H),1.39–1.29(m,6H).

[0092] Example 12 Synthesis of EGC-101 and EGC-201

[0093] The synthesis process for EGC-101 involves replacing intermediate 3 with intermediate 11 in the synthesis of EGC-1, while maintaining the same conditions as the synthesis of EGC-1. The synthesis of compound EGC-201 follows the same process as the hydrolysis of EGC-11 into EGC-211.

[0094] EGC-101, HRMS(ESI):m / z calcdforC 42 H 37 F2N2O 18 + :895.2004[M+H] + ;found:895.2005

[0095] EGC-201, 1HNMR (400MHz, DMSO-d6) δ11.11(s,1H),9.42–9.21(m,3H),8.77(s,2H),7.77(t,J=8.4Hz,1H) ,7.50(d,J=8.4Hz,1H),7.44(d,J=7.6Hz,1H),6.82(s,2H),6.41(s,2H),5.94(s,1H),5.84(s,1H),5.39( s,1H),5.08(dd,J=13.2,5.6Hz,1H),5.00(d,J=18.0Hz,1H),4.34(t,J=4.8Hz,2H),4.07(t,J=4.8Hz,2H ),3.83(dt,J=24.0,4.8Hz,4H),3.00–2.82(m,2H),2.78–2.56(m,3H),2.06–1.98(m,1H).HRMS(ESI):m / z calcd forC 39 H 36 F2N3O 17 + :856.2007[M+NH4] + ;found:856.2009

[0096] Example 13 Synthesis of EGC-102 and EGC-202

[0097] The synthesis process of EGC-102 involves replacing intermediate 3 with intermediate 11 in the synthesis of EGC-1, and replacing diethylene glycol bis-toluenesulfonate with triethylene glycol bis-toluenesulfonate. Other conditions are the same as in the synthesis of EGC-1. The synthesis of compound EGC-202 follows the same process as the hydrolysis of EGC-11 to EGC-211.

[0098] EGC-102, HRMS(ESI):m / z calcd forC 44 H 41 F2N2O 19 + 939.2266[M+H] + ;found:939.2268.

[0099] EGC-202, 1HNMR (400MHz, DMSO-d6) δ11.11 (s, 1H), 9.24 (s, 2H), 9.06 (s, 1H), 8.75 (s, 2H), 7.76 (dd, J = 8.8, 7.2Hz, 1H), 7. 50(d,J=8.4Hz,1H),7.43(d,J=7.2Hz,1H),6.81(s,2H),6.41(s,2H),5.93(d,J=2.3Hz,1H),5.83(d,J=2.3Hz,1H),5.39(t ,J=3.6Hz,1H),5.08(dd,J=12.8,5.6Hz,1H),4.97(s,1H),4.33(dd,J=6.0,3.6Hz,2H),4.02(dd,J=5.6,4.4Hz,2H),3.80( dd,J=5.6,3.6Hz,2H),3.66(t,J=4.8Hz,4H),3.62–3.58(m,2H),2.98–2.82(m,2H),2.71–2.52(m,3H),2.06–1.97(m,1H).

[0100] Example 14 Synthesis of EGC-103 and EGC-203

[0101] The synthesis process for EGC-103 involves replacing intermediate 3 with intermediate 11 in the synthesis of EGC-1, and replacing diethylene glycol bis-toluenesulfonate with tetraethylene glycol bis-toluenesulfonate. All other conditions are the same as in the synthesis of EGC-1. The synthesis of compound EGC-203 follows the same process as the hydrolysis of EGC-11 to EGC-211.

[0102] EGC-103, HRMS(ESI):m / z calcd forC 46 H 45 F2N2O 20 + :983.2528[M+H] + Found: 983.2529.

[0103] EGC-203, 1H NMR (400MHz, DMSO-d6) δ11.10(s,1H),9.30(s,1H),9.24(s,2H),8.73(s,2H),7.77(t,J=8. 0Hz,1H),7.47(dd,J=22.0,8.4Hz,2H),6.82(s,2H),6.41(s,2H),5.93(s,1H),5.83(s,1H), 5.39(s,1H),5.08(dd,J=12.4,5.2Hz,1H),4.97(s,1H),4.32(t,J=4.8Hz,2H),4.02(t,J=5. 2Hz,2H),3.88–3.43(m,12H),3.01–2.79(m,2H),2.75–2.52(m,3H),2.03(d,J=11.9Hz,1H).

[0104] Example 15 Synthesis of EGC-104 and EGC-204

[0105] The synthesis process for EGC-104 involves replacing intermediate 3 with intermediate 11 in the synthesis of EGC-1, and replacing diethylene glycol bis-toluenesulfonate with pentaethylene glycol bis-toluenesulfonate. All other conditions are the same as in the synthesis of EGC-1. The synthesis of compound EGC-204 follows the same process as the hydrolysis of EGC-11 to EGC-211.

[0106] EGC-204, 1H NMR (400MHz, DMSO-d6) δ11.10(s,1H),9.30(s,1H),9.24(s,2H),8.73(s,2H),7.77(t,J=8. 0Hz,1H),7.47(dd,J=22.0,8.4Hz,2H),6.82(s,2H),6.41(s,2H),5.93(s,1H),5.83(s,1H), 5.39(s,1H),5.08(dd,J=12.4,5.2Hz,1H),4.97(s,1H),4.32(t,J=4.8Hz,2H),4.02(t,J=5. 2Hz,2H),3.88–3.43(m,16H),3.01–2.79(m,2H),2.75–2.52(m,3H),2.03(d,J=11.9Hz,1H).

[0107] Example 16 Synthesis of EGC-105 and EGC-205

[0108] The synthesis process for EGC-105 involves replacing intermediate 3 with intermediate 11 in the synthesis of EGC-5, while maintaining the same conditions as the synthesis of EGC-5. The synthesis of compound EGC-205 follows the same process as the hydrolysis of EGC-11 to EGC-211.

[0109] EGC-205, 1HNMR (400MHz, DMSO-d6) δ11.17(s,1H),9.71(s,1H),9.54(s,2H),8.76(s,2H),8.46(d,J=8.4Hz,1H),8.09(d ,J=12.4Hz,2H),7.83(t,J=7.6Hz,1H),7.62(d,J=7.2Hz,1H),6.84(s,2H),6.41(s,2H),5.94(d,J=2.4Hz,1H),5.84(d, J=2.4Hz,1H),5.43–5.37(m,1H),5.15(dd,J=12.8,5.2Hz,1H),5.06(s,2H),4.98(s,1H),4.36(t,J=7.2Hz,2H),3.01–2 .84(m,2H),2.76–2.54(m,3H),2.49–2.43(m,2H),2.11–2.03(m,1H),1.89–1.79(m,2H),1.70–1.59(m,2H),1.28(m,2H).

[0110] Example 17 Synthesis of EGC-106 and EGC-206

[0111] The synthesis process for EGC-106 involves replacing intermediate 3 with intermediate 11 in the synthesis of EGC-5, and replacing 6-azidohexanoic acid pomadoumine with 8-azidooctanoic acid pomadoumine, while maintaining the same conditions as the synthesis of EGC-5. The synthesis of compound EGC-206 follows the same process as the hydrolysis of EGC-11 to EGC-211.

[0112] EGC-206, HNMR (400MHz, DMSO-d6): 11.17(s,1H),9.70(s,1H),9.53(s,2H),8.76(s,2H),8.47(d,J=8.0Hz,1H),8.08(d,J=3.2H z,2H),7.83(d,J=3.6Hz,1H),7.62(d,J=7.2Hz,1H),6.84(s,2H),6.41(s,2H),5.94(d,J=2.4Hz,1H),5.84(d,J=2.0Hz,1H),5.4 0(t,J=4.0Hz,1H),5.15(dd,J=12.8,5.6Hz,1H),5.06(s,2H),4.98(s,1H),4.33(t,J=7.2Hz,2H),2.99–2.84(m,2H),2.70–2.5 3(m,3H),2.46(t,J=7.6Hz,2H),2.12–2.03(m,1H),1.83–1.76(m,2H),1.65–1.56(m,2H),1.33–1.28(m,4H),1.23–1.15(m,2H).

[0113] Example 18 Synthesis of EGC-107 and EGC-207

[0114] The synthesis process for EGC-107 involves replacing intermediate 3 with intermediate 11 in the synthesis of EGC-5, and replacing 6-azidohexanoic acid pomadoumine with 10-azidodecanoic acid pomadoumine, while maintaining the same conditions as the synthesis of EGC-5. The synthesis of compound EGC-207 follows the same process as the hydrolysis of EGC-11 to EGC-211.

[0115] EGC-207, 1HNMR (400MHz, DMSO-d6) δ11.17(s,1H),9.70(s,1H),9.53(s,2H),8.75(s,2H),8.47(d,J=8.4Hz,1H),8.07(s ,2H),7.83(t,J=8.0Hz,1H),7.61(d,J=7.2Hz,1H),6.84(s,2H),6.41(s,2H),5.94(d,J=2.4Hz,1H),5.84(d,J=2.4Hz,1 H),5.43–5.37(m,1H),5.15(dd,J=12.8,5.2Hz,1H),5.07(s,2H),4.97(s,1H),4.32(t,J=6.8Hz,2H),3.01–2.84(m,2H) ,2.71–2.54(m,3H),2.46(t,J=7.6Hz,2H),2.11–2.02(m,1H),1.81–1.75(m,2H),1.66–1.57(m,2H),1.29–1.13(m,10H).

[0116] The structural formulas of intermediates 13 and 15 are as follows:

[0117]

[0118] Example 19 Synthesis of EGC-13 and EGC-213

[0119] The synthesis process for EGC-13 involves replacing intermediate 9 with intermediate 13 in the synthesis of EGC-11, while maintaining the same conditions as the synthesis of EGC-11. The synthesis of compound EGC-213 follows the same process as the hydrolysis of EGC-11 into EGC-211.

[0120] EGC-13, HRMS(ESI):m / z calcd forC 44 H 42 N3O 17 + 884.2509[M+NH4] + Found: 884.2510.

[0121] EGC-213, 1HNMR (400MHz, DMSO-d6) δ11.16(s,1H),9.70(s,1H),9.39–9.29(m,3H),9.06(s,1H),8.74(s,1H),8.46(d ,J=8.4Hz,1H),8.10(s,1H),8.05(s,1H),7.82(t,J=8.0Hz,1H),7.61(d,J=7.2Hz,1H),6.82(s,2H),6.40(s,2H),5. 93(d,J=2.4Hz,1H),5.82(d,J=2.4Hz,1H),5.38(s,1H),5.14(dd,J=12.8,5.2Hz,1H),4.96(s,1H),3.98–3.86(m,2H ),2.99–2.82(m,2H),2.71–2.54(m,3H),2.48–2.43(m,2H),2.10–2.02(m,1H),1.71–1.62(m,4H),1.49–1.41(m,2H).

[0122] Example 20: Synthesis of EGC-14 and EGC-214

[0123] The synthesis process for EGC-14 involves replacing intermediate 9 with intermediate 13 in the synthesis of EGC-11, and replacing pomadomine 6-bromohexanoate with pomadomine 8-bromooctanoate. All other conditions are the same as in the synthesis of EGC-11. The synthesis of compound EGC-214 follows the same process as the hydrolysis of EGC-11 to EGC-211.

[0124] EGC-14, HRMS(ESI):m / z calcdforC46H46N3O 16 + 896.2873[M+NH4] + ;found:896.2874.

[0125] EGC-214, 1HNMR (400MHz, DMSO-d6) δ11.17 (s, 1H), 9.70 (s, 1H), 9.32 (d, J = 5.6Hz, 3H), 9.07 (s, 1H), 8.75 (s, H), 8.65 (s, H),8.47(d,J=8.4Hz,1H),8.06(s,1H),7.83(t,J=8.0Hz,1H),7.61(d,J=7.2Hz,1H),6.83(s,2H),6.42(s,2H),5.94(d, J=2.4Hz,1H),5.84(d,J=2.0Hz,1H),5.44–5.34(m,1H),5.16(dd,J=12.8,5.6Hz,1H),4.98(s,1H),3.90(t,J=6.8Hz,2H ),3.01–2.83(m,2H),2.73–2.53(m,3H),2.46(t,J=7.6Hz,2H),2.12–2.02(m,1H),1.70–1.56(m,4H),1.39–1.29(m,6H).

[0126] Example 21 Synthesis of EGC-301 and EGC-401

[0127] The synthesis process for EGC-301 involves replacing intermediate 3 with intermediate 15 in the synthesis of EGC-1, while maintaining the same conditions as the synthesis of EGC-1. The synthesis of compound EGC-401 follows the same procedure as the hydrolysis of EGC-11 into EGC-211.

[0128] EGC-301, HRMS(ESI):m / z calcdforC 42 H 39 N2O 18 + 859.2193[M+H] + ;found:859.2194

[0129] EGC-401, δ11.10(s,1H),9.42–9.21(m,3H),9.09(s,1H),8.77(s,2H),8.10(s,1H),7.77(t,J=8.4Hz ,1H),7.50(d,J=8.4Hz,1H),7.44(d,J=7.6Hz,1H),6.82(s,2H),6.41(s,2H),5.94(s,1H),5.84(s,1H ),5.39(s,1H),5.08(dd,J=13.2,5.6Hz,1H),5.00(d,J=18.0Hz,1H),4.34(t,J=4.8Hz,2H),4.07(t, J=4.8Hz,2H),3.83(dt,J=24.0,4.8Hz,4H),3.00–2.82(m,2H),2.78–2.56(m,3H),2.06–1.98(m,1H).

[0130] Example 22 Synthesis of EGC-302 and EGC-402

[0131] The synthesis process for EGC-302 involves replacing intermediate 3 with intermediate 15 in the synthesis of EGC-1, and replacing diethylene glycol bis-toluenesulfonate with triethylene glycol bis-toluenesulfonate. All other conditions are the same as in the synthesis of EGC-1. The synthesis of compound EGC-402 follows the same process as the hydrolysis of EGC-11 to EGC-211.

[0132] EGC-302, HRMS(ESI):m / z calcdforC 44 H 43 N2O 19 + 903.2455[M+H] + ;found:903.2458.

[0133] EGC-402, 1H NMR(400MHz,DMSO-d6)δ11.11(s,1H),9.30(s,1H),9.24(s,2H),9.09(s,1H),8.89(s,1H),8.69(s,1H),8.07(s,1H),7.76(dd,J =8.8,7.2Hz,1H),7.50(d,J=8.4Hz,1H),7.43(d,J=7.2Hz,1H),6.81(s,2H),6.41(s,2H),5.93(d,J=2.3Hz,1H),5.83(d,J=2.3Hz ,1H),5.39(t,J=3.6Hz,1H),5.08(dd,J=12.8,5.6Hz,1H),4.95(s,1H),4.33(dd,J=6.0,3.6Hz,2H),4.02(dd,J=5.6,4.4Hz,2H) ,3.80(dd,J=5.6,3.6Hz,2H),3.66(t,J=4.8Hz,4H),3.62–3.58(m,2H),2.98–2.82(m,2H),2.71–2.52(m,3H),2.06–1.97(m,1H).

[0134] Example 23 Synthesis of EGC-303 and EGC-403

[0135] The synthesis process for EGC-303 involves replacing intermediate 3 with intermediate 15 in the synthesis of EGC-1, and replacing diethylene glycol bis-toluenesulfonate with tetraethylene glycol bis-toluenesulfonate. All other conditions are the same as in the synthesis of EGC-1. The synthesis of compound EGC-403 follows the same process as the hydrolysis of EGC-11 to EGC-211.

[0136] EGC-303, HRMS(ESI):m / z calcdforC 46 H 47 N2O 20 + 947.2717[M+H] + Found: 947.2719.

[0137] EGC-403, 1H NMR(400MHz,DMSO-d6)δ11.10(s,1H),9.30(s,1H),9.24(s,2H),9.05(s,1H),8.83(s,1H),8.61(s,1H ),8.04(s,1H),7.77(t,J=8.0Hz,1H),7.47(dd,J=22.0,8.4Hz,2H),6.82(s,2H),6.41(s,2H),5.93(s ,1H),5.83(s,1H),5.39(s,1H),5.08(dd,J=12.4,5.2Hz,1H),4.97(s,1H),4.32(t,J=4.8Hz,2H),4.0 2(t,J=5.2Hz,2H),3.88–3.43(m,12H),3.01–2.79(m,2H),2.75–2.52(m,3H),2.03(d,J=11.9Hz,1H).

[0138] Example 24 Synthesis of EGC-304 and EGC-404

[0139] The synthesis process for EGC-304 involves replacing intermediate 3 with intermediate 15 in the synthesis of EGC-1, and replacing diethylene glycol bis-toluenesulfonate with pentaethylene glycol bis-toluenesulfonate. All other conditions are the same as in the synthesis of EGC-1. The synthesis of compound EGC-404 follows the same process as the hydrolysis of EGC-11 to EGC-211.

[0140] EGC-304, HRMS(ESI):m / z calcdforC 48 H 51 N2O 21 + :991.2979[M+H] + ;found:991.2981

[0141] EGC-404, 1H NMR(400MHz,DMSO-d6)δ11.11(s,1H),9.31(s,1H),9.24(s,2H),9.05(s,1H),8.85(s,1H),8.62(s,1H ),8.04(s,1H),7.77(t,J=8.0Hz,1H),7.47(dd,J=22.0,8.4Hz,2H),6.83(s,2H),6.40(s,2H),5.94(s ,1H),5.83(s,1H),5.39(s,1H),5.08(dd,J=12.4,5.2Hz,1H),4.97(s,1H),4.32(t,J=4.8Hz,2H),4.0 2(t,J=5.2Hz,2H),3.88–3.43(m,16H),3.01–2.79(m,2H),2.75–2.52(m,3H),2.03(d,J=11.9Hz,1H).

[0142] Example 25 Synthesis of EGC-305 and EGC-405

[0143] The synthesis process for EGC-305 involves replacing intermediate 3 with intermediate 15 in the synthesis of EGC-5, while maintaining the same conditions as the synthesis of EGC-5. The synthesis of compound EGC-405 follows the same procedure as the hydrolysis of EGC-11 to EGC-211.

[0144] EGC-305, HRMS(ESI):m / z calcdforC 47 H 45 N6O 17 + 965.2836 [M+H] + Found: 965.2837

[0145] EGC-405, 1HNMR (400MHz, DMSO-d6) δ11.17(s,1H),9.71(s,1H),9.54(s,2H),9.32(s,1H),9.07(s,1H),8.86(s,1H),8.66(s,1H), 8.46(d,J=8.4Hz,1H),8.09(d,J=12.4Hz,2H),7.83(t,J=7.6Hz,1H),7.62(d,J=7.2Hz,1H),6.84(s,2H),6.41(s,2H),5.94(d,J=2 .4Hz,1H),5.84(d,J=2.4Hz,1H),5.43–5.37(m,1H),5.15(dd,J=12.8,5.2Hz,1H),5.06(s,2H),4.98(s,1H),4.36(t,J=7.2Hz,2H ),3.01–2.84(m,2H),2.76–2.54(m,3H),2.49–2.43(m,2H),2.11–2.03(m,1H),1.89–1.79(m,2H),1.70–1.59(m,2H),1.28(m,2H).

[0146] Example 26 Synthesis of EGC-306 and EGC-406

[0147] The synthesis process for EGC-306 involves replacing intermediate 3 with intermediate 15 in the synthesis of EGC-5, and replacing 6-azidohexanoic acid pomadoumine with 8-azidooctanoic acid pomadoumine, while maintaining the same conditions as the synthesis of EGC-5. The synthesis of compound EGC-406 follows the same procedure as the hydrolysis of EGC-11 to EGC-211.

[0148] EGC-306, HRMS(ESI): m / z calcd for C 49 H 49 N6O 17 + 993.3149[M+H] + ;found:993.3151

[0149] EGC-406, 1H NMR(400MHz,DMSO-d6)11.17(s,1H),9.70(s,1H),9.53(s,2H),9.32(s,1H),9.08(s,1H),8.84(s,1H),8.65(s ,1H),8.47(d,J=8.0Hz,1H),8.08(d,J=3.2Hz,2H),7.83(d,J=3.6Hz,1H),7.62(d,J=7.2Hz,1H),6.84(s,2H), 6.41(s,2H),5.94(d,J=2.4Hz,1H),5.84(d,J=2.0Hz,1H),5.40(t,J=4.0Hz,1H),5.15(dd,J=12.8,5.6Hz,1H) ,5.06(s,2H),4.98(s,1H),4.33(t,J=7.2Hz,2H),2.99–2.84(m,2H),2.70–2.53(m,3H),2.46(t,J=7.6Hz,2H).

[0150] Application Example 1: Effect of EGC-based glutamate dehydrogenase degrading agents on GDH activity

[0151] Add 65 μL of buffer C containing 2 mM EGCG to the first well of a 96-well plate (buffer contains 50 mM Tris, 0.003% Brij-35, 0.001% Tween 20, pH 8.0). Add 65 μL of buffer C to each of the remaining wells, and then dilute 2-fold. Next, add 65 μL of buffer C containing 0.3 mg / mL (or 2.5 mg / mL) GDH to each well and shake for 30 min. Finally, add 20 μL of a mixed assay reagent containing EZMTT: NADP. + (Final concentration: 150 μM), Glu (final concentration: 10 mM), EZMTT assay reagent (final concentration: 1×). The reaction was carried out under shaking conditions for 30 min at room temperature, and the OD values ​​during this time period were measured. 450 Numerical value, calculation IC 50 value.

[0152] The inhibitory activity curves of different concentration gradients and different types of degrading agents on glutamate dehydrogenase are shown in the figure below. Figure 1 As shown. By Figure 1 It is understood that although the degradative compound of the present invention has a larger structure, it can still directly inhibit GDH enzyme.

[0153] Application Example 2: GDH Degradation at the Cellular Level Using EGC-Based Glutamate Dehydrogenase Degrading Agents

[0154] Collect H22 cells in the logarithmic phase at a concentration of 1 × 10⁻⁶.5 Cells were seeded per well in 24-well plates and treated with different concentrations of glutamate dehydrogenase degrading agent for 24 h. Cells were digested with trypsin within 5 min, and the treated cells were collected. After centrifugation at 1000 rpm for 5 min, the supernatant was aspirated, and 50 μM RIPA lysis buffer containing glutamate dehydrogenase degrading agent was added. After incubation on ice for 30 min, the cells were centrifuged at 1000 rpm for 5 min, and the supernatant was collected. Protein concentration was determined using the BCA method. Based on the protein concentration, the total protein loading amount was 40 μg, ensuring a loading volume of 20 μL. 5 μL of 5×SDS-PAGE protein loading buffer was added and mixed thoroughly. The cells were then heated at 95 °C for 10 min to denature the protein. Load 5 μL of protein molecular weight standard marker and 20 μL of denatured protein sample onto an SDS-PAGE gel for electrophoresis analysis. Transfer the protein from the gel to a PVDF membrane. After transfer, carefully remove the PVDF membrane with tweezers, cut off a corner of the front side of the membrane for marking, and soak it in 5% skim milk. Block the membrane on a shaker at room temperature for 2 hours. Incubate with primary antibody (mouse-derived anti-GDH and anti-GAPDH) overnight at 4°C. The next day, remove the PVDF membrane, recover the primary antibody dilution, and wash. Incubate with secondary antibody (goat anti-mouse) at room temperature for 1.5 hours. After incubation, wash three times with PBST solution for 10 minutes each time. For colorimetric development, equal volumes of detection solutions A and B from the ECL chemiluminescence ultrasensitivity kit were mixed in advance and prepared for later use. After removing excess PBST solution from the membrane, the ECL chemiluminescence detection solution was added dropwise. After incubation in the dark for 1 min, images were taken and analyzed using the Bio-Rad Chemi-Doc MP imaging system, and quantitative analysis was performed using Image Lab v.5.2 software (Bio-Rad Laboratories). The results of the degradation of GDH and GAPDH at the cellular level by the EGC-based glutamate dehydrogenase degrader are as follows: Figure 2 As shown. By Figure 2 It can be seen that, at the cellular level, the degrading agent prepared in this invention can degrade GDH, but the 100 μmol / L control 3 (EGC) did not show significant degradation activity. The compounds synthesized in this invention all exhibited a certain degree of degradation under 100 μM conditions, and some compounds (e.g., ECG-1 to EGC-7, EGC-11 to EGC-16) showed significant GDH degradation activity while also degrading GAPDH.

[0155] Application Example 3: Antitumor Cell Activity of EGC-Based Glutamate Dehydrogenase Degraders

[0156] H22 cells in logarithmic growth phase were prepared into cell suspensions using RPMI 1640 complete medium and seeded into 96-well plates (3 × 10⁶ cells / well). 3Cells / well (volume 150 μL) were incubated in a humidified 5% CO2, 37°C cell culture incubator for 4 h. After cell attachment, 50 μL of a series of compound solutions (compound solutions using DMSO as solvent, final concentration: 0–10 μM, trifold dilution) were added to each well. Control 1 was prepared by adding 0.05% DMSO to each well in RPMI 1640 complete medium. Cell-free wells were used as control 1. Each test concentration was performed in triplicate. Cells were cultured at 37°C for 5 days, followed by incubation of 20 μL of EZMTT assay reagent in each well for 4 h. Absorbance at 450 nm was read every h using a microplate reader, and the inhibition rate was calculated. The inhibitory activity curve of the EGC-based glutamate dehydrogenase degrader against H22 tumor cells is shown in the figure. Figure 3 As shown, by Figure 3 It is known that the compounds of the present invention all have certain antitumor activity and distinct active regions, which originate from the critical point of GDH enzyme degradation.

[0157] Application Example 4: Protective effect of EGC-based glutamate dehydrogenase degrading agents on glutamate-damaged PC12 cells

[0158] PC12 cells in the logarithmic growth phase were seeded into 96-well plates (3 × 10⁶ cells / well) using RPMI 1640 complete medium. 3 Cells / well (volume 150 μL) were incubated in a humidified 5% CO2, 37°C cell culture incubator for 4 h. After cell attachment, 50 μL of RPMI 1640 complete medium with 6 mM glutamate and 50 μL of RPMI 1640 complete medium without glutamate were added, along with 100-fold diluted DMSO solutions of the test compounds (final concentration: 0–90 μM, tertiary dilutions). A control group was prepared with 0.05% DMSO in RPMI 1640 complete medium. Cell-free wells served as blank controls. Each test concentration was performed in triplicate. Cells were cultured at 37°C for 1–5 days, with daily observation of cell growth. 20 μL of EZMTT assay reagent was added to detect viable cells, and absorbance at 450 nm was read using a microplate reader to calculate the inhibition rate. The effect of different concentrations of glutamate dehydrogenase degrading agents on the viability of PC12 cells treated with different concentrations of glutamate is as follows. Figure 4 As shown, the relative survival rates of PC12 cells treated with different compounds at a glutamate concentration of 15 mM were analyzed using a one-way ANOVA test. Compared with the control group (0 mM group): *p < 0.05, **p < 0.01, ***p < 0.001. Figure 4It is evident that the degrading agent of the present invention has a stronger toxicity reduction ability compared to the control EGC and EGCG. Moreover, at high concentrations, the control EGC and EGCG exhibit toxicity, while the degrading agent of the present invention is non-toxic. This provides an effective approach for the treatment of HHS genetic diseases.

[0159] The degradation effects of adding 30 μM and 100 μM glutamate dehydrogenase degrading agent, EGC, EGCG, and ECG on intracellular GDH protein, respectively, and the cytotoxicity of glutamate to PC12 cells when adding 3 μM and 100 μM glutamate dehydrogenase degrading agent, EGC, EGCG, and ECG are shown in Table 1.

[0160] Table 1. Degradation of intracellular GDH protein and cytotoxic effects of glutamate on PC12 cells.

[0161]

[0162]

[0163] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A glutamate dehydrogenase-degrading agent based on EGC, characterized in that, By linking EGC-like compounds with PROTAC-like small molecules, a glutamate dehydrogenase-based degrading agent is formed. The degrading agent contains the following general structural formula: Where X is independent, it is O or NH; Y and Z are independent, they are F or H; and L is... n1 is 1, 2, 3 or 4, n2 is 2, 3, 4 or 5, n3 is 5, 7 or 9, and n4 is 5, 7 or 9.

2. The glutamate dehydrogenase degrading agent according to claim 1, characterized in that, Compounds with general structural formula I include those with the following structural formulas:

3. The glutamate dehydrogenase degrading agent according to claim 1, characterized in that, Compounds of general structural formula II include those with the following structures:

4. The glutamate dehydrogenase degrading agent according to claim 1, characterized in that, Compounds of general structural formula III include those with the following structures:

5. The use of the EGC-based glutamate dehydrogenase degrading agent according to any one of claims 1 to 4 in the preparation of a drug for degrading GDH protein.

6. The application according to claim 5, characterized in that, EGC-2, EGC-3, EGC-12, and EGC-15 are used to prepare drugs that degrade the GDH and GAPDH dual proteins.

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