Preparation method and application of biphenyl tetrazole derivative

Abstract

The invention relates to the field of medicine, in particular to a preparation method of biphenyl tetrazole derivatives and application of the biphenyl tetrazole derivatives in preparation, prevention or treatment of neurodegenerative diseases and related diseases caused by plateau hypoxia, and the structure of the biphenyl tetrazole derivatives is shown as a formula 8 X in the specification, and R3 is selected from hydrogen, fluorine, chlorine, iodine, methyl, methoxyl, dimethylamino, trifluoromethyl, hydroxyl, carboxyl, nitryl, methoxycarbonyl, acrylate and acrylamide. The biphenyl tetrazole derivative prepared in the invention can destroy Nrf2-Keap1 protein-protein interaction (PPI), and has a very good protection effect on tested cell models of hydrogen peroxide and 6-hydroxydopamine nerve injury and cobalt chloride hypoxia injury. Experimental data show that the biphenyl tetrazole derivative disclosed by the invention is a potential therapeutic molecule, and a certain basis is provided for research and development of drugs.

Description

Technical Field

[0001] The present invention discloses a preparation method of a class of biphenyltetrazolyl derivatives and their use in the preparation, prevention or treatment of neurodegenerative diseases and related diseases caused by plateau hypoxia, belonging to the field of medicine. Background Art

[0002] Neurodegenerative diseases and diseases caused by acute hypoxia at high altitude may be closely related to the Nrf2-Keap1 signaling system in the body.

[0003] Neurodegenerative diseases result from the progressive loss of neurons or myelin, leading to cognitive or physical dysfunction. As of 2024, the number of patients worldwide has exceeded 50 million, of which Alzheimer's disease (AD) is the most common, and Parkinson's disease (PD) ranks second. Forecasts show that by 2025, the number of PD patients worldwide will reach 25.2 million, posing a serious threat to health. The pathogenesis of such diseases (such as AD, PD, and multiple sclerosis MS) is largely related to oxidative stress, inflammatory response, and protein homeostasis imbalance in the body. Therefore, the Nrf2-Keap1 system, which plays a core role in regulating these key pathological mechanisms, has become an important target for the treatment of neurodegenerative diseases.

[0004] Acute hypoxia at high altitude can trigger a range of health problems, among which high-altitude cerebral edema (HACE) is a typical and severe condition that severely impacts cognitive function. Rapidly reaching high altitudes causes a sudden drop in atmospheric oxygen partial pressure, leading to insufficient oxygen supply, which can trigger acute mountain sickness (AMS), the most advanced form of which is HACE. The core manifestation of HACE is significant cognitive impairment, including severe headaches, extreme fatigue, confusion, disorientation, memory loss, poor concentration, impaired judgment, and even hallucinations or coma. Its pathological basis involves hypoxia-induced severe oxidative stress (generating large amounts of reactive oxygen species (ROS)), activation of the inflammatory response, disruption of the blood-brain barrier, and neuronal damage and death. In this process, the Nrf2-Keap1 system plays a key role in endogenous antioxidant defense. Normally, the Keap1 protein anchors the transcription factor Nrf2 in the cytoplasm and promotes its degradation. However, the severe oxidative stress induced by acute hypoxia at high altitude disrupts the binding of Keap1 to Nrf2, stabilizing Nrf2 and allowing its translocation to the nucleus. Within the cell nucleus, Nrf2 binds to specific DNA sequences (antioxidant response elements, AREs), initiating the transcriptional expression of a series of downstream antioxidant enzymes and phase II detoxification enzymes (such as HO-1, NQO1, SOD, and GSH), aiming to eliminate excess ROS, mitigate oxidative damage, and protect neurons and vascular endothelial cells. Therefore, effective activation of the Nrf2 pathway is considered a key protective mechanism against high-altitude hypoxia-induced neuroinflammation and cognitive impairment.

[0005] Although tetrazole drugs are widely used in the field of neuroprotection, the development of tetrazole drugs targeting the Nrf2-Keap1 system still faces challenges. There are currently no reports on the effects of biphenyl tetrazole compounds on the nervous system and the treatment of high-altitude hypoxia-related diseases. Summary of the Invention

[0006] In the first aspect of the present invention, a method for preparing a class of biphenyl tetrazole derivatives and their use in the preparation, prevention, or treatment of neurodegenerative diseases and related diseases caused by high-altitude hypoxia are provided. Secondly, the provided biphenyl tetrazole derivatives provide novel, previously unreported targets for their use in the preparation, prevention, or treatment of neurodegenerative diseases and related diseases caused by high-altitude hypoxia. Disruption of the Nrf2-Keap1 protein-protein interaction (PPI) is a novel target for molecules with this structural chemistry to exert the aforementioned pharmacological activity.

[0007] The embodiment of the present invention is achieved as follows:

[0008] The preparation and application of a class of biphenyltetrazolyl derivatives described in the present invention have the structural formula X as follows:

[0009]

[0010] Preferably, R1, R2, and R3 are selected from hydrogen, fluorine, chlorine, iodine, methyl, methoxy, dimethylamino, trifluoromethyl, hydroxy, carboxyl, nitro, methoxycarbonyl, acrylate, and acrylamide.

[0011] Specifically, the biphenyl tetrazole derivative having the ability to disrupt Nrf2-Keap1 protein-protein interaction (PPI) is selected from the following compounds:

[0012]

[0013] In a second aspect of the present invention, there is provided a method for preparing the biphenyltetrazolyl derivatives described in the first aspect, comprising the following steps:

[0014] Compounds LY-1~LY-8 and YL-1~YL-8 were obtained by Suzuki coupling, hydrolysis, 1,3-dipolar cycloaddition, nucleophilic substitution and other reactions.

[0015]

[0016] a) 1,4-dioxane, aqueous potassium carbonate solution, tetrakis(triphenylphosphine)palladium, phenylborate, 100°C, 3h; b) aqueous hydrogen peroxide solution, potassium hydroxide, DMSO, rt, 30min; c) 4-methylpyridine, DPPA, 140-160°C, overnight; d) acetonitrile, DIPEA, dimethylcarbamoyl chloride, 30°C, overnight.

[0017] 具体地,所述制备方法如下:

[0018] 中间体的通用合成方法:

[0019] 合成中间体B1~B8:

[0020] Weigh potassium carbonate (564 mg, 2.0 eq) and dissolve it in 4 mL of water (Solvent A). Add 4-bromobenzeneacetonitrile (400 mg, 1.0 eq), phenylboronate (1.5 eq), and tetrakis(triphenylphosphine)palladium (235 mg, 0.1 eq) to a flask. Add 1,4-dioxane (16 mL) and Solvent A (4 mL). Heat to 95°C and reflux for 2-4 hours under nitrogen. After completion of the reaction, extract, mix, and purify by column chromatography (PE:EA = 12:1-8:1) to obtain compounds B1-B8 in 85%-94% yields.

[0021] 合成中间体C1~C8和M2:

[0022] Intermediates B1-B8 (400 mg, 1.0 eq) were dissolved in DMSO (3 mL / mmol). Potassium hydroxide (1.4 eq) was added, and 30% H₂O₂ (1.0 mL) was slowly added dropwise. The reaction was allowed to proceed at RT for 30 min under nitrogen. After completion of the reaction, 5-10 times the amount of ice water was added to adjust the pH to 8. The mixture was stirred and filtered to obtain C1-C8 in 75%-87% yields.

[0023] Dissolve 4-biphenylacetonitrile (1.0 g, 1.0 eq) in DMSO (16 mL), add potassium hydroxide (406.5 mg, 1.4 eq), and slowly add 30% H₂O₂ (2.5 mL) dropwise. React at rt for 30 min under nitrogen. After the reaction, add 5-10 times the amount of ice water, adjust the pH to 8, and filter with stirring to obtain a pale yellow solid with a yield of 89.6%.

[0024] 合成中间体D1~D8和M3:

[0025] Intermediates C1-C8 (200 mg, 1.0 eq) were added to a 25 mL flask, followed by 4-methylpyridine (2.0 mL) and diphenylphosphoryl azide (2.5 eq). The mixture was heated to 140°C and refluxed under nitrogen overnight. After completion of the reaction, the mixture was diluted with 15 mL of water and the pH was adjusted to 4-6. The mixture was extracted, stirred, and purified by column chromatography (DCM:MeOH = 80:1-60:1) to obtain D1-D8 in 61%-82% yields.

[0026] The intermediate M2 (322 mg, 1.0 eq) was added to a 25 mL flask, followed by the addition of 4-methylpyridine (3.2 mL), diphenyl phosphorazide (0.82 mL, 2.5 eq), and the reaction was carried out at 140°C under reflux with nitrogen protection overnight. After the reaction was completed, 15 mL of water was added for dilution, the pH was adjusted to 4-6, and extraction, sample mixing, and column chromatography purification were carried out to obtain M3 white solid 325 mg with a yield of 90.3%.

[0027] In a third aspect of the present application, a pharmaceutical preparation is provided, which comprises the above-mentioned biphenyl tetrazole derivative or the above-mentioned pharmaceutical composition, and a pharmaceutically acceptable carrier and / or adjuvant. The preparation is selected from oral preparations and parenteral administration preparations, and can be tablets, pills, capsules or injections.

[0028] In a fourth aspect of the present application, the use of the above-mentioned biphenyl tetrazole derivative or its pharmaceutically acceptable salt, composition in the preparation of an antitumor drug is provided.

[0029] In one or more embodiments, the use includes the preparation, prevention or treatment of neurodegenerative diseases and related diseases caused by high altitude hypoxia.

[0030] In vitro neuroprotective and anti-hypoxia studies show that the biphenyl tetrazole derivative disclosed in the present application has good protective effect on the tested cell models of hydrogen peroxide and 6-hydroxydopamine nerve injury, cobalt chloride hypoxia injury and hypoxia injury. BRIEF DESCRIPTION OF DRAWINGS

[0031] The drawings accompanying the specification of the present application serve to provide a further understanding of the present application, and the illustrative embodiments of the present application and their descriptions serve to explain the present application, and do not constitute an improper limitation on the present application.

[0032] Figure 1 Figure 1 is a docking diagram of the test compounds LY-3(A), LY-3(B) and LY-3(C) with the Keap1-Kelch domain.

[0033] Figure 2 Figure 2 is the toxicity of the test compounds to PC 12 cells.

[0034] Figure 3 Figure 3 is the neuroprotective activity of the test compounds on PC 12 cells induced by H2O2 to cause oxidative damage.

[0035] Figure 4 Figure 4 is the protective activity of the test compounds on PC 12 cells induced by hypoxia to cause oxidative damage.

[0036] Figure 5 Figure 5 is the protective activity of the test compounds on PC 12 cells induced by cobalt chloride to cause oxidative damage.

[0037] Figure 6 Figure 3: Effects of the test compounds on the expression of antioxidant enzymes in damaged PC 12 cells.

[0038] Figure 7 The test compound alleviates oxidative damage dependent on Nrf2 activation.

[0039] Figure 8 The test compound is formulated as follows: DETAILED DESCRIPTION

[0040] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0041] Example 1:

[0042]

[0043] D1 (1.0 eq) was suspended in acetonitrile (4.9 mL) and dissolved in DIPEA (0.2 mL, 2.5 eq). Dimethylaminoformyl chloride (0.08 mL, 2.0 eq) was added and allowed to react overnight at rt under nitrogen. After the reaction, the mixture was extracted and purified by preparative thin-layer chromatography (DCM:EA = 220:1) to obtain LY-1 and YL-2.

[0044] The reaction is shown in reaction formula 1

[0045]

[0046] LY-1, yield 9.5%; 1 H NMR(400MHz,Chloroform-d)δ7.49(d,J=7.6Hz,4H),7.32(d,J=7.9Hz,

[0047] 2H),7.11(t,J=8.7Hz,2H),4.47(s,2H),3.08(s,3H),2.74(s,3H); 13C NMR (101MHz, CDCl3) δ162.77,160.31,154.72,146.84,138.59,135.37,135.34,132.25,128 .50,127.56,127.48,126.30,114.84,114.62,37.86,36.46,28.59; MS(ESI)m / zcalculated for C 17 H 16 FN5O[2M+Na] + 673.2576, found 673.1669.

[0048] YL-1, yield 26.7%; 1 H NMR(400MHz,Chloroform-d)δ7.53–7.48(m,4H),7.42(d,J=8.0Hz,2H),

[0049] 7.11(t,J=8.6Hz,2H),4.37(s,2H),3.26(s,3H),3.12(s,3H); 13 C NMR (101MHz, CDCl3) δ164.09,162.65,160.20,146.75,138.12,135.77,135.73,133.9 0,128.37,127.60,127.52,126.35,114.71,114.50,38.21,37.14,30.34; MS(ESI)m / z calculated forC 17 H 16 FN5O[M+Na] + 348.1237, found 348.0603.

[0050] Example 2:

[0051]

[0052] D2 (1.0 eq) was suspended in acetonitrile (2.4 mL) and dissolved in DIPEA (0.35 mL, 2.5 eq). Dimethylcarbamoyl chloride (0.08 mL, 2.0 eq) was added and the mixture was heated to 40°C overnight under nitrogen. After completion of the reaction, the mixture was extracted and purified by preparative thin-layer chromatography (DCM:EA = 220:1) to obtain LY-2 and YL-2.

[0053] LY-2, yield 11.2%; 1H NMR(400MHz,Chloroform-d)δ8.28(d,J=8.3Hz,2H),7.70(d,J=8.4Hz,

[0054] 2H),7.59(d,J=7.9Hz,2H),7.42(d,J=7.8Hz,2H),4.51(s,2H),3.13(s,3H),2.88(s,3H); 13 C NMR(101MHz,CDCl3)δ154.61,146.79,146.16,145.68,137.08,134.13,128.84,126.75,126.67,123.17,38.12,36.66,28.75,28.67;MS(ESI)m / z calculated forC 17 H 16 N6O3[M+H] + 353.1317,found 353.2027.

[0055] YL-2,产率为18.9%; 1 H NMR(400MHz,Chloroform-d)δ8.29(d,J=8.8Hz,2H),7.71(d,J=8.8Hz,

[0056] 2H),7.59(d,J=8.2Hz,2H),7.49(d,J=8.2Hz,2H),4.40(s,2H),3.26(s,3H),3.13(s,3H); 13 C NMR(101MHz,CDCl3)δ164.63,147.52,146.87,137.47,136.58,129.57,127.60,127.50,123.94,39.06,38.00,31.23;MS(ESI)m / z calculated for C 17 H 16 N6O3[M+Na] + 375.1182,found 375.0599.

[0057] 实施例3:

[0058]

[0059] D3 (1.0 eq) was suspended in acetonitrile (3.2 mL) and dissolved dropwise with DIPEA (0.25 mL, 2.5 eq). Dimethylcarbamoyl chloride (0.1 mL, 2.0 eq) was added and allowed to react overnight at rt under nitrogen. After completion of the reaction, the mixture was extracted and purified by preparative thin-layer chromatography (DCM:EA = 90:1) to obtain LY-3 and YL-3.

[0060] LY-3, yield 16.8%; 1 H NMR(400MHz,Chloroform-d)δ7.32–7.20(m,7H),7.15(d,J=6.8Hz,1H),

[0061] 4.48(s,2H),3.07(s,3H),2.69(s,3H),2.23(s,3H); 13 C NMR (101MHz, CDCl3) δ155.56,147.70,141.22,140.78,134.88,132.55,130.22,12 9.42,128.59,127.33,125.69,38.52,37.20,29.44,20.25; MS(ESI)m / zcalculated for C 18 H 19 N5O[M+Na] + 344.1488,found344.0930.

[0062] YL-3, yield 10.2%; 1 H NMR(400MHz,Chloroform-d)δ7.40(d,J=7.6Hz,2H),7.32–7.18(m,6H),

[0063] 4.39(s,2H),3.27(s,3H),3.14(s,3H),2.27(s,3H); 13 C NMR (101MHz, CDCl3) δ164.93,147.58,141.13,140.54,135.10,134.13,130.10,1 29.53,129.36,128.38,127.07,125.55,39.04,37.95,31.21,20.27; MS(ESI)m / z calculated for C 18 H 19 N5O[M+Na] + 344.1488,found 344.0930.

[0064] Example 4:

[0065]

[0066] D4 (1.0 eq) was suspended in acetonitrile (2.0 mL) and dissolved dropwise with DIPEA (0.14 mL, 2.5 eq). Dimethylcarbamoyl chloride (0.06 mL, 2.0 eq) was added and allowed to react overnight at rt under nitrogen. After completion of the reaction, the product was extracted and purified by preparative thin-layer chromatography (DCM:EA = 220:1) to obtain LY-4 and YL-4.

[0067] LY-4, yield 30.0%; 1 H NMR(400MHz,Chloroform-d)δ7.54(d,J=7.4Hz,2H),7.33(dd,J=17.0,8.2

[0068] Hz,5H),7.17(d,J=7.1Hz,1H),4.47(s,2H),3.06(s,3H),2.67(s,3H),2.41(s,3H); 13 C NMR (101MHz, CDCl3) δ155.55,147.66,140.54,139.98,138.31,132.88,131.75,130.54,1 29.20,128.59,128.14,127.52,127.26,123.86,38.55,37.19,29.39,21.33; MS(ESI)m / z calculated for C 18 H 19 N5O[M+Na] + 344.1488,found 344.0960.

[0069] YL-4, yield 34.0%; 1 H NMR(400MHz,Chloroform-d)δ7.55(d,J=7.8Hz,2H),7.44–7.29(m,5H),

[0070] 7.16(d,J=7.3Hz,1H),4.37(s,2H),3.25(s,3H),3.11(s,3H),2.41(s,3H); 13CNMR (101MHz, CDCl3) δ165.27,147.89,140.74,140.32,138.44,134.90,131.96,130.75,129.3 7,128.77,128.16,127.93,127.61,124.24,39.33,38.25,31.49,21.63;MS(ESI)m / zcalculated for C 18 H 19 N5O[M+Na] + 344.1488, found 344.0957.

[0071] Example 5:

[0072]

[0073] D5 (1.0 eq) was suspended in acetonitrile (1.0 mL) and dissolved in DIPEA (0.08 mL, 2.5 eq). Dimethylcarbamoyl chloride (0.04 mL, 2.0 eq) was added and the mixture was heated to 30°C overnight under nitrogen. After completion of the reaction, the mixture was extracted and purified by preparative thin-layer chromatography (DCM:EA = 220:1) to obtain LY-5 and YL-5.

[0074] LY-5, yield 26.6%; 1 H NMR(400MHz,Chloroform-d)δ7.38(d,J=8.0Hz,2H),7.30(d,J=7.9Hz,

[0075] 2H),7.16(d,J=7.9Hz,2H),7.11(s,1H),7.09(s,1H),4.32(s,2H),2.90(s,3H),2.52(s,3H),2.24(s,3H); 13 CNMR(101MHz, CDCl3)δ154.73,146.85,139.51,136.45,136.29,131.86,128.57,128.41,126.21,125.76,37.73,36.36,28.57,20.07;

[0076] MS (ESI) m / z calculated for C 18 H 19 N5O[M+Na] + 344.1488, found 344.0935.

[0077] YL-5, yield 64.4%;1 H NMR(400MHz,Chloroform-d)δ7.54(d,J=7.8Hz,2H),7.47(d,J=7.8Hz,

[0078] 2H),7.41(d,J=7.8Hz,2H),7.24(d,J=7.8Hz,2H),4.36(s,2H),3.25(s,3H),3.11(s,3H),2.39(s,3H); 13 CNMR(101MHz, CDCl3)δ164.17,146.77,139.01,136.74,136.07,133.55,128.46,128.26,126.28,125.84,38.21,37.13,30.36,20.06; MS(ESI)m / zcalculated for C 18 H 19 N5O[M+Na] + 344.1488,found 344.0942.

[0079] Example 6:

[0080]

[0081] D6 (100 g, 1.0 eq) was suspended in acetonitrile (2.3 mL) and dissolved in DIPEA (0.16 mL, 2.5 eq). Dimethylcarbamoyl chloride (0.07 mL, 2.0 eq) was added and the mixture was heated to 30°C overnight under nitrogen. After completion of the reaction, the mixture was extracted and purified by preparative thin-layer chromatography (DCM:EA = 90:1) to yield LY-6 and YL-6.

[0082] LY-6, yield 27.8%; 1 H NMR(400MHz,Chloroform-d)δ7.40(d,J=7.7Hz,2H),7.25–7.15(m,4H),

[0083] 6.94(dd,J=21.1,7.7Hz,2H),4.40(s,2H),3.72(s,3H),2.97(s,3H),2.54(s,3H); 13C NMR (101 MHz, CDC13) δ 155.31, 154.66, 146.86, 137.08, 131.60, 129.58, 128.94, 128.70, 127.93, 127.67, 119.86, 110.15, 54.40, 37.54, 36.18, 28.63; MS (ESI) m / z calculated for C 18 H 19 N5O2[M+Na] + 360.1436, found 360.0581.

[0084] YL-6, yield 16.7%; 1 H NMR (400 MHz, Chloroform-d) δ 7.42 (d, J = 7.9 Hz, 2H), 7.32 (d, J = 7.9 Hz,

[0085] 2H), 7.25 - 7.18 (m, 2H), 6.98 - 6.87 (m, 2H), 4.29 (s, 2H), 3.73 (s, 3H), 3.18 (s, 3H), 3.05 (s, 3H); 13 C NMR (101 MHz, CDC13) δ 164.15, 155.37, 146.79, 136.35, 133.41, 129.73, 129.06, 128.88, 127.65, 127.52, 119.79, 110.11, 54.46, 38.22, 37.12, 30.44; MS (ESI) m / z calculated for C 18 H 19 N5O2[M+Na] + 360.1436, found 360.0594.

[0086] Example 7:

[0087]

[0088] D7 (100 mg, 1.0 eq) was suspended in acetonitrile (2.3 mL), DIPEA (0.16 mL, 2.5 eq) was added dropwise, dimethylcarbamoyl chloride (0.07 mL, 2.0 eq) was added, and the reaction was carried out at 30°C overnight under nitrogen protection. After the reaction was completed, extraction and purification by preparative thin layer chromatography (DCM:EA = 220:1) were carried out to obtain LY-7 and LY-7

[0089] LY-7, yield 23.2%; 1H NMR(400MHz,Chloroform-d)δ7.54(d,J=7.8Hz,2H),7.33(t,J=8.3Hz,

[0090] 3H),7.17–7.04(m,2H),6.90(d,J=8.3Hz,1H),4.48(s,2H),3.85(s,3H),3.06(s,3H),2.70(s,3H).; 13 C NMR(101MHz,CDCl3)δ158.96,154.70,146.83,140.72,139.43,132.36,128.88,128.42,126.48,118.41,111.86,111.71,54.29,37.77,36.39,28.59;MS(ESI)m / z calculated for C 18 H 19 N5O2[M+Na] + 360.1436,found 360.0600.

[0091] YL-7,产率为24.6%; 1 H NMR(400MHz,Chloroform-d)δ7.53(s,2H),7.38(d,J=34.2Hz,3H),7.12

[0092] (d,J=25.5Hz,2H),6.89(s,1H),4.37(s,2H),3.85(s,3H),3.25(s,3H),3.10(s,3H); 13 C NMR(101MHz,CDCl3)δ164.09,158.87,146.75,141.14,138.93,134.05,128.74,128.28,126.51,118.51,111.75,111.64,54.25,38.19,37.11,30.35;MS(ESI)m / zcalculated for C 18 H 19 N5O2[M+Na] + 360.1436,found 360.0597.

[0093] 实施例8:

[0094]

[0095] D8 (1.0 eq) was suspended in acetonitrile (3.7 mL) and dissolved in DIPEA (1.0 mL, 2.5 eq). Dimethylcarbamoyl chloride (0.12 mL, 2.0 eq) was added and the mixture was heated to 30°C overnight under nitrogen. After completion of the reaction, the mixture was extracted and purified by preparative thin-layer chromatography (DCM:EA = 220:1) to obtain LY-8 and YL-8.

[0096] LY-8, yield 16.8%; 1 H NMR (400MHz, Chloroform-d) δ7.49 (s, 4H), 7.29 (d, J = 7.9Hz, 2H), 6.96 (d,

[0097] J=8.3Hz,2H),4.46(s,2H),3.84(s,3H),3.05(s,3H),2.67(s,3H); 13 C NMR (101MHz, CDCl3) δ158.33,154.75,146.86,139.17,131.66,131.51,128. 41,126.95,125.95,113.27,54.34,37.74,36.36,28.67,28.54; MS(ESI)m / z calculated forC 18 H 19 N5O2[M+Na] + 360.1436,found 360.0825.

[0098] YL-8, yield 28.0%; 1 H NMR(400MHz,Chloroform-d)δ7.56–7.45(m,4H),7.40(d,J=7.9Hz,2H),

[0099] 6.96(d,J=8.2Hz,2H),4.35(s,2H),3.84(s,3H),3.25(s,3H),3.11(s,3H).; 13 CNMR(101MHz, CDCl3)δ164.19,158.11,146.76,138.67,133.21,132.14,128.27,127.02,126.04,113.16,54.30,38.21,37.12,30.33; MS(ESI)m / z calculated for C 18 H 19 N5O2[M+Na] + 360.1436,found 360.0848.

[0100] Example 9:

[0101] This example is used to determine the cell proliferation activity of the compounds LY-1 to LY-8 and YL-1 to YL-8 of the present invention on PC12 cells. The protocol is as follows: cells were seeded at a concentration of 5,000 cells / well in a 96-well transparent plate, with 100 μL per well. The cells were cultured for 12 hours at 5% CO2 and 37°C. Then, a series of compound solutions (100 μL) prepared in serum-free medium were added to the wells and cultured for another 24 hours. Cell viability was assessed using the MTT assay. A microplate reader was set up and the absorbance at 570 nm was measured using the microplate reader. The results were analyzed after they were obtained. Cell viability (%) = (OD drug group - OD blank control) / (OD negative control - OD blank control). GraphPad Prism 8.0 was used to plot the results.

[0102] The MTT experiment found that the test compound of the present invention has low cytotoxicity, and the cell survival rate is higher than 75% under the conditions of 1-100 μM. It is very suitable for development for the preparation, prevention or treatment of neurodegenerative diseases and related diseases caused by high-altitude hypoxia.

[0103] Example 10:

[0104] This example is used to determine the protective effect of the compounds of the present invention on PC12 cells damaged by hydrogen peroxide in vitro. The protocol is as follows: PC12 cells were plated at 8×10 3 Cells were seeded at a density of 100 μg / well in a 96-well plate and cultured for 24 hours. After the cells adhered, various concentrations of the test compound of the present invention were added for 24 hours according to the experimental design. The remaining medium was aspirated and replaced with DMEM medium containing 500 μM H₂O₂ until the cell damage was approximately 50%. Cell viability was determined using the MTT assay described above.

[0105] In the above hydrogen peroxide injury experiment, compared with the H2O2 group, some of the test compounds of the invention, such as LY-1, LY-3, and LY-5, increased cell survival rate. The above experimental results indicate that the compounds of the present invention have certain neuroprotective potential.

[0106] Example 11:

[0107] This example was used to determine the protective effect of the compounds of the present invention against hypoxic PC12 cells in vitro. The protocol was as follows: Weigh the test compound of the present invention, dissolve it in DMSO, and prepare a 50 mM stock solution for future use. This solution was then diluted with culture medium to the desired concentration (μM) and prepared immediately before use. The prepared test compound was added to a 96-well plate seeded one day in advance (seed plate size based on the desired cell count), and negative and blank controls were set up on the 96-well plate. After 24 hours of drug exposure, the 96-well plate was placed in a tri-gas incubator (1% O₂ + 5% CO₂ + 94% N₂) for 24 hours. 10 μL of MTT was added to each well and allowed to incubate for an additional 4 hours. When blue-purple formazan crystals were observed, 100 μL of the tri-gas solution was added to each well and allowed to incubate overnight (for at least 8 hours). A microplate reader was used to measure absorbance at 570 nm, and the results were analyzed. Cell viability (%) = (OD drug group - OD blank control) / (OD negative control - OD blank control).

[0108] In the above in vitro hypoxic injury experiment, compared with the hypoxic injury group, some of the test compounds of the invention increased the cell survival rate. The above experimental results indicate that the compounds of the present invention have certain anti-hypoxia potential.

[0109] Example 11:

[0110] This example was used to determine the protective effect of the compounds of the present invention on PC12 cells damaged by hypoxia in vitro (cobalt chloride model). This example was used to determine the protective effect of the compounds of the present invention on PC12 cells damaged by hypoxia in vitro. The protocol was as follows: Weigh the test compound of the present invention, dissolve it in DMSO, and prepare a 50 mM stock solution for future use. This solution was then diluted with culture medium to the desired concentration (μM) and prepared immediately before use. The prepared test compound was added to a 96-well plate seeded one day in advance (depending on the desired cell count), and negative and blank controls were set up on the 96-well plate. After 24 hours of drug exposure, the old culture medium was aspirated and DMEM medium containing 400 μM CoCl₂ was added to the wells for incubation until the cell damage reached approximately 60%. Cell viability was then determined using the MTT assay described above. A microplate reader was used to measure absorbance at 570 nm, and the results were analyzed. Cell viability (%) = (OD drug group - OD blank control) / (OD negative control - OD blank control).

[0111] In the above in vitro hypoxic injury experiment, compared with the CoCl2 injury group, some of the test compounds of the invention increased the cell survival rate. The above experimental results indicate that the compounds of the present invention have certain anti-hypoxia potential.

[0112] Example 12:

[0113] This example was used to determine the effects of the compounds described herein on the expression of antioxidant enzymes or proteins. The protocol was as follows: cells were treated with the compounds described herein for 24 hours and then damaged with H₂O₂ or 6-OHDA. After treatment, cells were harvested and lysed with RIPA buffer. Protein content was determined using the Bradford assay, and intracellular levels of NQO1, TrxR1, Trx1 activity, total thiol groups, and total GSH were measured.

[0114] After treatment with the compound of the present invention, the activities of NQO1, Trx1, and TrxR1 and the GSH content are significantly increased, alleviating the damage caused by H2O2 or 6-OHDA treatment, indicating that the compound of the present invention increases the expression of antioxidant molecules in cells and enhances the antioxidant capacity.

[0115] Example 12:

[0116] This example was used to determine whether Nrf2 is responsible for the neuroprotective effects of the compounds described herein. siRNA interference was used to reduce Nrf2 expression in cells, reducing Nrf2 expression levels in PC12-siNrf2 cells to approximately 50% of that in PC12siNC cells. Oxidative damage experiments were then performed on the transfected cells. Compared to PC12 siNC cells, the protective effects of the compounds described herein on PC12-siNrf2 cells were significantly reduced, indicating that Nrf2 activation is crucial for the neuroprotective and anti-hypoxic effects of the compounds described herein.

Claims

1. The preparation of a class of biphenyltetrazolyl derivatives described in the present invention, and their use in the preparation, prevention or treatment of drugs for neurodegenerative diseases and high-altitude acute hypoxia-related diseases, wherein the structural formula X is: Preferably, R1, R2, and R3 are selected from hydrogen, fluorine, chlorine, iodine, methyl, methoxy, dimethylamino, trifluoromethyl, hydroxy, carboxyl, nitro, methoxycarbonyl, acrylate, and acrylamide.

2. The biphenyltetrazolyl derivative according to claim 1, wherein The compound used in the preparation, prevention or treatment of neurodegenerative diseases and plateau acute hypoxia-related diseases is selected from the following compounds:

3. The method for preparing the biphenyltetrazolyl derivative according to any one of claims 1 to 2, characterized in that: The steps include: Compounds LY-1~LY-8 and YL-1~YL-8 were obtained by Suzuki coupling, hydrolysis, 1,3-dipolar cycloaddition, nucleophilic substitution and other reactions.

4. The preparation method according to claim 3, wherein The general synthesis methods of intermediates include Suzuki coupling, hydrolysis, 1,3-dipolar cycloaddition, nucleophilic substitution and other reaction processes.

5. A pharmaceutical preparation, characterized in that The invention comprises the biphenyltetrazolyl derivative according to any one of claims 1 to 2, and a pharmaceutically acceptable carrier and / or excipient.

6. Use of the biphenyltetrazolyl derivative or pharmaceutically acceptable salt thereof or the composition according to any one of claims 1 to 2 in the preparation of antitumor drugs.

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