A class of 3-hydroxypyrazine-2-carboxylic acid dextromethorphan or fenchol ester derivatives and uses thereof

Abstract

The application discloses a kind of 3-hydroxy-pyrazine-2-methanoic acid dextromethorphan or fenchol ester derivatives and purposes thereof, the derivative has as shown in formula (I) structure.The experimental results show that the compound of the application can significantly inhibit lipopolysaccharide-induced RAW264.7 cell inflammatory response, reduce the release of TNF-alpha and IL-1 beta and other inflammatory factors, and can improve the nerve dysfunction of stroke model mice after injury, and show good anti-inflammatory and nerve function repair effect.The compound of the application can be used for preparing medicaments for treating stroke, cerebral ischemia-reperfusion injury, Alzheimer's disease and other neuroinflammatory related diseases, and has the application value of promoting nerve function recovery.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically relating to a class of derivatives of 3-hydroxypyrazine-2-carboxylic acid dextranol or fentanyl ester and their use in the treatment of stroke, Alzheimer's disease and other neuroinflammatory-related diseases. Background Technology

[0002] Stroke is an acute cerebrovascular disease caused by impaired cerebral blood circulation, leading to localized cerebral ischemia, hypoxia, or hemorrhage. It is characterized by high morbidity, high mortality, high disability rate, and high recurrence rate, making it a serious threat to human health (Lancet Neurol., 2021, 20: 795-820; Circulation, 2017, 135: 759-771). Ischemic stroke accounts for more than 80% of all strokes. In recent years, with the development of reperfusion therapy techniques such as intravenous thrombolysis and mechanical thrombectomy, the acute mortality rate of stroke patients has significantly decreased. However, a large number of patients still suffer from neurological sequelae such as motor dysfunction, cognitive impairment, and mood disorders (N. Engl. J. Med., 2023, 388: 887-906). Therefore, developing novel drugs that can promote the recovery of neurological function after stroke has become an important research direction in the field of stroke treatment.

[0003] Numerous studies have shown that neuroinflammatory response is one of the important pathological mechanisms of secondary brain injury after stroke (Nat. Med., 2011, 17: 796-808). Following cerebral ischemia, microglia, infiltrating macrophages, and astrocytes are activated, releasing various inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), thereby exacerbating neuronal damage, blood-brain barrier disruption, and neural network dysfunction (J. Neuroinflammation, 2019, 16: 142). Therefore, regulating the neuroinflammatory response after stroke is considered an important therapeutic strategy for promoting neurological function recovery.

[0004] Alzheimer's disease (AD) is a common neurodegenerative disease characterized by β-amyloid deposition, abnormal phosphorylation of tau protein, and persistent neuroinflammatory responses (Nat. Rev. Neurol., 2021, 17: 157-172). Recent studies have shown that abnormal microglial activation and the sustained release of pro-inflammatory cytokines such as TNF-α and IL-1β are important mechanisms leading to neuronal damage and cognitive impairment. Neuroinflammation not only participates in the development of Alzheimer's disease but is also closely related to various other neurodegenerative diseases such as Parkinson's disease. Therefore, inhibiting neuroinflammation has become an important direction in the development of drugs for neurological diseases (Lancet Neurol., 2015, 14: 388-405; Signal Transduct Target Ther. 2023, 8: 267).

[0005] Therefore, developing novel candidate compounds that combine anti-inflammatory activity and neuroprotective effects is of great significance for the research and development of drugs for the treatment of nervous system diseases. Summary of the Invention

[0006] The purpose of this invention is to provide a class of 3-hydroxypyrazine-2-carboxylic acid dextranol or fumarate derivatives, which have anti-inflammatory effects and promote the recovery of neurological function after stroke, and can be used to prepare drugs for the treatment of stroke, cerebral ischemia-reperfusion injury, Alzheimer's disease and other neuroinflammatory-related diseases.

[0007] The technical solution of the present invention is as follows:

[0008] This invention provides a class of 3-hydroxypyrazine-2-carboxylic acid dextranol or fumarate ester derivatives or pharmaceutically acceptable salts thereof, the chemical structure of which is shown in formula (I):

[0009] ;

[0010] Where: R 1 R 2 Each is independently an -H or C1-C6 alkyl group;

[0011] R 3 for or ;

[0012] R 4 For -H, or R 5 It is a C1-C8 alkyl group; R 6 It is a C1-C8 alkyl group or a C3-C6 cycloalkyl group.

[0013] As a preferred implementation scheme, R 1 and R 2 Each is an alkyl group, either -H or C1-C3, more specifically, R. 1 and R 2 Each can be either -H or methyl.

[0014] As another preferred implementation, R 1 and R 2 Selected from C1-C3 alkyl groups, more preferably specific ones, R 1 and R 2 It is -CH3.

[0015] As a preferred implementation, R³ is .

[0016] As a preferred implementation, R³ is .

[0017] As a preferred implementation scheme, R 4 For H.

[0018] As another preferred implementation, R 4 for R 6 It is selected from acetyl, propionyl, butyryl, isobutyryl, pivaloyl or cyclohexyl acyl.

[0019] As another preferred implementation, R 4 for R 5 It is selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl.

[0020] Furthermore, the derivatives described in this invention are selected from any one or more of the following compounds:

[0021]

[0022]

[0023] .

[0024] The present invention also provides a pharmaceutical composition comprising the 3-hydroxypyrazine-2-carboxylic acid dextranol or fentanyl ester derivative thereof as described herein, and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0025] The present invention also provides the use of the 3-hydroxypyrazine-2-carboxylic acid dextranol or fentanyl ester derivative thereof or a pharmaceutically acceptable salt thereof or the composition thereof in the preparation of a medicament for inhibiting an inflammatory response.

[0026] The present invention also provides the use of the 3-hydroxypyrazine-2-carboxylic acid dextranol or fentanyl ester derivative thereof or a pharmaceutically acceptable salt thereof or the composition thereof in the preparation of medicaments for neuroinflammatory diseases.

[0027] The neuroinflammatory diseases described in this invention include, but are not limited to, stroke, cerebral ischemia-reperfusion injury, Alzheimer's disease, and other neuroinflammatory diseases.

[0028] The beneficial effects of this invention are:

[0029] This invention provides a novel class of 3-hydroxypyrazine-2-carboxylic acid dextranol or frankincide ester derivatives. Experimental results show that the compounds of this invention can significantly inhibit lipopolysaccharide-induced inflammatory responses in RAW264.7 cells, reduce the release of inflammatory factors such as TNF-α and IL-1β, and exhibit good anti-inflammatory activity. Furthermore, the compounds of this invention can improve neurological dysfunction after stroke in mouse models, demonstrating good potential for promoting post-stroke neurological function recovery.

[0030] Therefore, the compounds described in this invention possess both anti-inflammatory and neuroprotective activities, and can be used to prepare drugs for treating stroke, cerebral ischemia-reperfusion injury, and neuroinflammatory-related diseases, showing promising application prospects in the field of post-stroke neurological function repair. Attached Figure Description

[0031] Figure 1 Different doses of target compound 1 were administered to mice 5–11 days after stroke, and the dose-response relationship was evaluated by the fall rate in a grid test on day 12.

[0032] Figure 2 Target compounds 1, 5, and 11 were administered to mice 5-11 days after stroke, and the pharmacodynamics of the target compounds were compared using a grid test on day 12 based on the fall rate.

[0033] Figure 3 The target compounds 1, 5, and 11 were administered to 5-month-old 5×FAD mice for one month, and the novel object discrimination index was measured to evaluate the efficacy of the target compounds in improving cognitive impairment. Detailed Implementation

[0034] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0035] Example 1: Synthesis of the target compound

[0036] Synthesis of target compound 1:

[0037] Synthesis of 3-hydroxy-5,6-dimethylpyrazine-2-carboxylic acid:

[0038]

[0039] Aminomalonamide (6.80 g, 58.08 mmol) was dissolved in a mixture of methanol and water. An appropriate amount of aqueous sodium hydroxide solution was slowly added under ice bath conditions, followed by dropwise addition of 2,3-butanedione (5.0 g, 58.08 mmol). After stirring for 10 min, the temperature was raised to room temperature or a suitable temperature to continue the reaction. After the reaction was complete as monitored by TLC, water was added to quench the reaction. The pH of the system was adjusted to slightly acidic with 2 M dilute hydrochloric acid. The precipitated solid was collected by filtration and dried under vacuum to obtain 3-hydroxy-5,6-dimethylpyrazine-2-carboxamide (intermediate 1).

[0040] Intermediate 1 was added to an excess of 10% sodium hydroxide aqueous solution and heated at 80 °C for 12 h. After the reaction was complete (the system was clear) by TLC monitoring, the temperature was lowered to room temperature. 2 M dilute hydrochloric acid was slowly added dropwise to adjust the pH to 2-3, at which point a large amount of solid precipitated. The mixture was filtered, the filter cake was washed with a small amount of cold water, and dried under vacuum to obtain 3-hydroxy-5,6-dimethylpyrazine-2-carboxylic acid (intermediate 2).

[0041]

[0042] 3-Hydroxy-5,6-dimethylpyrazine-2-carboxylic acid (intermediate 2) (0.50 g, 2.98 mmol) was dissolved in 10 mL of dichloromethane (DCM), followed by the addition of dextranol (0.46 g, 2.98 mmol), the condensing agent N,N'-dicyclohexylcarbodiimide (DCC, 0.92 g, 4.47 mmol), and the esterification catalyst 4-dimethylaminopyridine (DMAP, 0.11 g, 0.89 mmol). The mixture was stirred overnight at room temperature. After the reaction was complete, the white insoluble residue was removed by filtration, and the filtrate was washed successively with dilute hydrochloric acid, water, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether / ethyl acetate v / v = 200:1) to give dextranol ester of 3-hydroxy-5,6-dimethylpyrazine-2-carboxylic acid (target compound 1) in 55% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.25 (s, 1H), 4.99 (ddd, J =10.0, 3.5, 1.9 Hz, 1H), 2.32 (ddd, J = 10.0, 6.0, 3.0 Hz, 1H), 2.25 (s, 3H), 2.22 (s, 3H), 1.99 (ddd, J = 12.4, 9.0, 4.0 Hz, 1H), 1.67 (dq, J = 10.7, 3.6Hz, 2H), 1.26 – 1.14 (m, 2H), 1.01 (dd, J = 13.7, 3.5 Hz, 1H), 0.88 (s, 3H),0.83 (s, 3H), 0.81 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 165.36, 155.37,80.59, 49.29, 48.04, 44.82, 36.65, 28.13, 27.15, 20.03, 19.54, 19.15, 18.63,13.94.

[0043] Synthesis of 3-hydroxy-5,6-dimethylpyrazine-2-carboxylic acid dextranol ester (target compound 2): Following the synthesis method of 3-hydroxy-5,6-dimethylpyrazine-2-carboxylic acid dextranol ester (target compound 1), it was prepared from intermediate 3-hydroxy-5,6-dimethylpyrazine-2-carboxylic acid (intermediate 2) and dextranol. 1 H NMR (400 MHz, DMSO-d6) δ 4.42 (d, J =1.9 Hz, 1H), 2.23 (d, J = 12.8 Hz, 6H), 1.75 (d, J = 14.7 Hz, 1H), 1.68 (d, J= 3.8 Hz, 1H), 1.61 (d, J = 10.5 Hz, 2H), 1.46 – 1.34 (m, 1H), 1.26 – 1.11(m, 2H), 1.08 (s, 3H), 1.02 (s, 3H), 0.75 (s, 3H). 13 C NMR (101 MHz, DMSO-d6)δ 165.50, 155.41, 87.08, 48.59, 48.31, 33.88, 30.04, 26.71, 25.99, 24.98,20.67, 19.73, 19.64, 18.66.

[0044] Synthesis of 3-hydroxypyrazine-2-carboxylate dextranol ester (target compound 3): Following the synthetic method of 3-hydroxy-5,6-dimethylpyrazine-2-carboxamide (intermediate 1), intermediate 3-hydroxypyrazine-2-carboxylic acid was prepared. Following the synthetic method of 3-hydroxy-5,6-dimethylpyrazine-2-carboxylate dextranol ester (target compound 1), it was prepared from intermediate 3-hydroxypyrazine-2-carboxylic acid and dextranol. 1 H NMR (400 MHz, DMSO-d6) δ 7.66 (s, 1H), 7.43 (s, 1H), 5.02 (dt, J = 10.1, 2.7 Hz, 1H), 2.37 – 2.28 (m, 1H), 1.92 (ddd, J = 12.6, 9.3,4.2 Hz, 1H), 1.66 (p, J = 4.4 Hz, 2H), 1.26 – 1.14 (m, 2H), 1.01 (dd, J =13.7, 3.5 Hz, 1H), 0.88 (s, 3H), 0.83 (s, 3H), 0.81 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 82.16, 50.65, 49.42, 46.07, 37.78, 29.39, 28.41, 21.33, 20.43,15.20.

[0045] Synthesis of target compound 4: It was prepared by referring to the synthesis method of 3-hydroxypyrazine-2-carboxylic acid dextranol ester (target compound 3), using intermediate 3-hydroxypyrazine-2-carboxylic acid and ferrous alcohol as raw materials. 1 H NMR (400 MHz, DMSO-d6) δ 7.65(d, J = 5.0 Hz, 1H), 7.44 (s, 1H), 4.44 (d, J = 2.1 Hz, 1H), 1.72 – 1.67 (m,2H), 1.61 (d, J = 10.9 Hz, 2H), 1.44 – 1.37 (m, 1H), 1.19 (d, J = 5.3 Hz, 2H), 1.08 (d, J = 1.9 Hz, 3H), 1.03 (d, J = 2.0 Hz, 3H), 0.76 (d, J = 1.9 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 81.98, 50.59, 49.35, 46.08, 37.84, 35.16,29.40, 28.42, 26.28, 23.85, 21.31, 20.43, 15.23.

[0046] Synthesis of target compound 5:

[0047] Synthesis route:

[0048]

[0049] Procedure: Target compound 1 (1.0 g, 5.95 mmol) was dissolved in anhydrous THF, and DMAP (0.85 g, 0.60 mmol) was added as an acid-binding agent. The mixture was cooled in an ice bath. Propionyl chloride (0.68 g, 7.14 mmol) was slowly added dropwise, and the mixture was gradually brought to room temperature with stirring after the addition was complete. The reaction was quenched with water after TLC monitoring until complete. The separated organic phase was washed successively with dilute hydrochloric acid, saturated sodium bicarbonate aqueous solution, and saturated brine. After drying with anhydrous sodium sulfate and concentration under reduced pressure, the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate volume ratio = 250:1) to prepare target compound LK-43-1 in 30% yield. 1 HNMR (400 MHz, DMSO-d6) δ 7.65 (d, J = 5.0 Hz, 1H), 7.44 (s, 1H), 4.44 (d, J =2.1 Hz, 1H), 1.73 – 1.67 (m, 2H), 1.61 (d, J = 10.9 Hz, 2H), 1.43 – 1.36 (m,1H), 1.19 (d, J = 5.3 Hz, 2H), 1.08 (d, J = 1.9 Hz, 3H), 1.03 (d, J = 2.0 Hz,3H), 0.76 (d, J = 1.9 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 172.58, 163.76,156.23, 151.65, 150.83, 133.52, 81.35, 49.31, 48.15, 44.75, 36.56, 28.19,27.43, 27.10, 22.08, 21.62, 20.05, 19.14, 13.93, 8.98.

[0050] Synthesis of target compound 6: It was prepared by referring to the synthesis method of target compound 5, using target compound 1 and butyryl chloride as raw materials. 1 H NMR (400 MHz, DMSO-d6) δ 5.02 (dt, J = 10.0, 2.8 Hz, 1H), 2.58(t, J = 7.3 Hz, 2H), 2.52 (s, 3H), 2.50 (s, 3H), 2.34 (ddt, J = 14.0, 9.9,4.0 Hz, 1H), 1.93 (dt, J = 13.2, 5.4 Hz, 1H), 1.74 – 1.65 (m, 2H), 1.60 (p, J= 7.4 Hz, 2H), 1.32 – 1.23 (m, 1H), 1.22 – 1.12 (m, 1H), 0.99 (dd, J = 13.8,3.5 Hz, 1H), 0.93 (t, J = 7.4 Hz, 3H), 0.88 (s, 3H), 0.83 (s, 3H), 0.79 (s,3H). 13 C NMR (101 MHz, DMSO-d6) δ 171.68, 163.76, 156.20, 151.61, 150.79,133.59, 81.33, 49.31, 48.14, 44.76, 36.58, 35.66, 28.19, 27.11, 22.10, 21.63,20.04, 19.14, 18.01, 13.93, 13.82.

[0051] Synthesis of target compound 7: It was prepared by referring to the synthesis method of target compound 5, using target compound 1 and butyryl chloride as raw materials. 1H NMR (400 MHz, DMSO-d6) δ 5.01 (ddd, J = 10.0, 3.5, 2.1 Hz, 1H), 2.82 (p, J = 7.0 Hz, 1H), 2.36 – 2.28 (m, 1H), 1.93 (ddd, J = 13.2, 9.1, 3.8Hz, 1H), 1.74 – 1.65 (m, 2H), 1.27 (ddd, J = 11.7, 9.4, 3.4 Hz, 1H), 1.21 (s,3H), 1.19 (s, 3H), 1.16 (dt, J = 10.1, 3.4 Hz, 1H), 1.01 – 0.97 (m, 1H), 0.88(s, 3H), 0.83(s, 3H), 0.79(s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 174.85,163.78, 156.18, 151.53, 150.90, 133.69, 81.29, 49.36, 48.16, 44.76, 36.55,33.85, 28.17, 27.11, 22.08, 21.65, 20.06, 19.15, 18.85, 13.96.

[0052] Synthesis of target compound 8: It was prepared by referring to the synthesis method of target compound 5, using target compound 1 and pivaloyl chloride as raw materials. 1 H NMR (400 MHz, DMSO-d6) δ 5.14 – 4.91 (m, 1H), 2.52 (s, 3H), 2.50 (s, 3H), 2.32 (dd, J = 8.8, 4.9 Hz, 1H), 1.94 (ddd, J = 12.9, 9.1, 3.9Hz, 1H), 1.77 – 1.61 (m, 2H), 1.26 (s, 9H), 1.23 – 1.14 (m, 2H), 1.01 (dd, J= 13.7, 3.5 Hz, 1H), 0.88 (s, 3H), 0.83 (s, 3H), 0.80 (s, 3H). 13C NMR (101MHz, DMSO-d6) δ 162.41, 79.80, 48.01, 46.78, 43.37, 35.12, 31.13, 26.76,25.72, 20.67, 20.29, 18.66, 17.76, 12.60.

[0053] Synthesis of target compound 9: It was prepared by referring to the synthesis method of target compound 5, using target compound 1 and cyclohexylformyl chloride as raw materials. 1 H NMR (400 MHz, DMSO-d6) δ 5.03 – 4.98 (m, 1H), 2.52 (s, 3H), 2.49 (s, 3H), 2.17 – 2.09 (m, 1H), 1.95 (d, J = 12.3 Hz, 2H), 1.77 – 1.66 (m,5H), 1.25 (dt, J = 26.6, 7.5 Hz, 9H), 0.99 (dd, J = 13.7, 3.5 Hz, 1H), 0.88(s, 3H), 0.83 (s, 3H), 0.79 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 81.28, 48.16,44.77, 42.72, 42.51, 36.57, 29.18, 28.71, 28.17, 25.94, 25.44, 25.15, 21.65,20.06, 19.16, 13.95.

[0054] Synthesis of target compound 10: It was prepared by referring to the synthesis method of target compound 5, using target compound 1 and itaconic acid monomethyl ester chloride as raw materials. 1H NMR (400 MHz, DMSO-d6) δ 6.53 (s, 1H), 6.17 (s, 1H), 5.01 (d, J = 10.0 Hz, 1H), 3.58 (d, J = 2.5 Hz, 3H), 2.53 (s, 3H), 2.51 (s,3H), 2.38 – 2.28 (m, 1H), 1.91 – 1.83 (m, 1H), 1.65 (d, J = 4.9 Hz, 2H), 1.23– 1.13 (m, 2H), 0.96 (d, J = 13.8 Hz, 1H), 0.88 – 0.85 (m, 3H), 0.81 (s, 3H),0.75 (d, J = 2.5 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 175.56, 169.04, 168.37,160.97, 156.70, 155.36, 138.52, 138.06, 137.61, 86.22, 57.10, 54.02, 52.91,49.49, 41.86, 41.19, 32.87, 31.88, 26.85, 26.36, 24.79, 23.87, 18.66.

[0055] Synthesis of target compound 11: It was prepared by referring to the synthesis method of target compound 5, using target compound 1 and itaconic acid monooctyl ester chloride as raw materials. 1H NMR (400 MHz, Chloroform-d) δ 5.18 (ddd, J = 9.8, 3.4,2.1 Hz, 1H), 4.06 (d, J = 16.5 Hz, 1H), 3.46 (s, 1H), 2.51 (d, J = 2.8 Hz,6H), 2.44 (dt, J = 10.0, 4.2 Hz, 1H), 2.25 – 2.18 (m, 1H), 1.92 (dd, J =12.8, 4.0 Hz, 2H), 1.81 (dq, J = 12.1, 4.3 Hz, 1H), 1.75 (d, J = 4.6 Hz, 1H),1.72 – 1.65 (m, 3H), 1.42 – 1.37 (m, 2H), 1.36 – 1.30 (m, 5H), 1.25 (d, J =12.2 Hz, 5H), 1.19 (dd, J = 14.0, 3.5 Hz, 2H), 0.96 (s, 3H), 0.91 (s, 6H),0.90 – 0.75 (m, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 169.39, 160.53, 156.82,82.62, 49.39, 49.25, 48.14, 44.99, 36.60, 34.04, 31.53, 30.90, 30.26, 29.80,28.07, 27.12, 25.69, 25.04, 22.79, 22.44, 21.31, 19.81, 18.99, 13.73, 1.12.

[0056] Example 2: The inhibitory effect of the target compound on the increase of inflammatory factors in RAW264.7 mouse monocytes and macrophages induced by lipopolysaccharide (LPS).

[0057] Mouse monocyte-macrophage RAW264.7 cells were used as an in vitro inflammation evaluation model. RAW264.7 cells were routinely cultured in high-glucose DMEM medium containing 10% fetal bovine serum and incubated at 37 ℃ in a 5% CO2 incubator. Cells in the logarithmic growth phase were harvested at a rate of 1×10⁻⁶ cells / cells. 5Cells were seeded at a concentration of 500 μL / well in 24-well plates, and after adhesion, the medium was changed and the cells were grouped. The experiment included a normal culture group, an LPS model group (incubated with 1 μM LPS alone for 24 h), a positive control group (pretreated with 1 μM dexamethasone for 4 h followed by incubation with 1 μM LPS for 24 h), and a target compound treatment group (pretreated with 2 μM target compound for 1 h followed by incubation with 1 μM LPS for 24 h). After co-incubation, the cell culture supernatant from each group was collected by centrifugation, and the expression levels of inflammatory factors were quantitatively analyzed using an IL-1β and TNF-α ELISA kit (Jiangsu Kaiji Biotechnology Co., Ltd.). All procedures were strictly performed according to the kit instructions. After the colorimetric reaction was terminated, the absorbance at the dominant wavelength of 450 nm was measured using a multi-mode microplate reader (570 nm for IL-1β and 540 nm for TNF-α as dual-wavelength calibration references). Based on the standard curves plotted using the standards, the absolute expression concentrations of IL-1β and TNF-α in each sample were calculated. Experimental data are expressed as mean ± standard error and statistical analysis was performed using GraphPad Prism 10.0 software.

[0058] Table 1. Effects of the target compound on the expression concentrations of TNF-α and IL-1β in a lipopolysaccharide-induced cellular inflammation model (n=3)

[0059] ; The results are shown in Table 1. LPS stimulation significantly increased the expression levels of TNF-α and IL-1β in RAW264.7 cells, showing a significant difference compared to the normal control group, indicating the successful establishment of the inflammation model. Compared to the LPS model group, target compounds 1–4 all reduced the expression levels of inflammatory factors to varying degrees. Among them, target compound 1 showed the most significant inhibitory effect on TNF-α and IL-1β, followed by target compound 2; target compounds 3 and 4 also exhibited certain anti-inflammatory activities.

[0060] The above results indicate that the 3-hydroxypyrazine-2-carboxylic acid dextranol or fumarate derivatives of the present invention can effectively inhibit LPS-induced inflammatory responses, reduce the release of inflammatory factors, and have good anti-inflammatory activity.

[0061] Based on the anti-inflammatory activity evaluation results in Example 2 and the structural characteristics of the target compounds, representative target compounds 1, 5 and 11 were selected for further pharmacodynamic evaluation in animal models of stroke.

[0062] Example 3: Dose-response relationship of the effect of target compound 1 on improving post-stroke motor function in a mouse PT model.

[0063] A focal cerebral ischemia model was created by inducing the production of oxygen free radicals from rose red under light, which damage vascular endothelial cells. Adult mice were anesthetized with 2% isoflurane gas, fixed on a stereotactic frame, and their skulls were exposed through a midline scalp incision. Connective tissue was removed and the skulls dried. A cold light source with a 2 mm diameter luminescent spot was vertically placed above the skull, with its center offset 1.5 mm to the right from the anterior fontanelle. After an intraperitoneal injection of rose red (100 mg / kg) for 5 min, the intensity of the cold light source was adjusted to 12000 Lux and irradiated for 15 min. After irradiation, the cold light source was removed, and the area was sutured and disinfected. Throughout the procedure, the animals' body temperature was maintained at 37 ± 0.5 ºC using a temperature-controlled infrared lamp. The sham-operated group was anesthetized, had their scalp cut open, and were injected with rose red, but did not receive cold light irradiation. After the surgery, the animals were returned to their respective cages and carefully monitored until they regained consciousness. Behavioral tests were conducted on mice placed on a grid device with parameters of length × width × height: 32 × 20 × 50 cm and a grid size of 1.2 × 1.2 cm at one week before stroke modeling, on day 4 after modeling, and on day 12 after modeling. From day 5 to 11 after modeling, compound 1 was administered intraperitoneally (ip) daily at doses of 1, 2, 4, and 8 mg / kg, respectively. A camera was placed below the grid to record the entire walking process. The animals were allowed to walk freely on the grid for 5 minutes, and the number of missteps per foot and the total number of steps were counted. The percentage of missteps per foot was calculated using the formula: [number of missteps / (number of missteps + number of non-missteps) × 100]. A misstep was defined as the animal's limb crossing the grid without support or merely the wrist resting on the edge of the grid.

[0064] The results are as follows Figure 1 As shown, the grid test failure rates were comparable and low in all groups before modeling. On day 4 after PT modeling, the grid test failure rates were comparable in all groups but significantly higher than before modeling, indicating successful establishment of the stroke model. After one week of continuous administration, compared with the solvent group, the grid test failure rate in the target compound 1 treatment group was significantly reduced. Furthermore, within the dose range of (1 mg / kg - 4 mg / kg), the grid test failure rate decreased with increasing dose of target compound 1, indicating that target compound 1 can significantly improve motor dysfunction in the mouse PT model, and this effect shows a clear dose-response relationship.

[0065] The above results indicate that the target compound 1 of the present invention can significantly improve stroke-induced motor dysfunction and promote the repair of neurological function after stroke.

[0066] Example 4: Effects of target compounds 1, 5, 11 on improving post-stroke motor function in a mouse PT model.

[0067] A focal cerebral ischemia model was created by inducing the production of oxygen free radicals from rose red under light, which damage vascular endothelial cells. Adult mice were anesthetized with 2% isoflurane gas, fixed on a stereotactic frame, and their skulls were exposed through a midline scalp incision. Connective tissue was removed and the skulls dried. A cold light source with a 2 mm diameter luminescent spot was vertically placed above the skull, with its center offset 1.5 mm to the right from the anterior fontanelle. After an intraperitoneal injection of rose red (100 mg / kg) for 5 min, the intensity of the cold light source was adjusted to 12000 Lux and irradiated for 15 min. After irradiation, the cold light source was removed, and the area was sutured and disinfected. Throughout the procedure, the animals' body temperature was maintained at 37 ± 0.5 ºC using a temperature-controlled infrared lamp. The sham-operated group was anesthetized, had their scalp cut open, and were injected with rose red, but did not receive cold light irradiation. After the surgery, the animals were returned to their respective cages and carefully monitored until they regained consciousness. Behavioral tests were conducted on mice placed on a grid device with parameters of length × width × height: 32 × 20 × 50 cm and a grid size of 1.2 × 1.2 cm at one week before stroke modeling, on day 4 after modeling, and on day 12 after modeling. From day 5 to 11 after modeling, mice were administered 4 mg / kg of the target compound (compound ip) daily, for compounds 1, 5, and 11, respectively. A camera was placed below the grid to record the entire walking process. The animals were allowed to walk freely on the grid for 5 minutes, and the number of missteps per foot and the total number of steps were counted. The percentage of missteps per foot was calculated using the formula: [number of missteps / (number of missteps + number of non-missteps) × 100]. A misstep was defined as the animal's limb crossing the grid without support or merely the wrist resting on the edge of the grid.

[0068] The results are as follows Figure 2 As shown, compared with the sham-operated group, the grid test failure rate in the solvent group was significantly increased, indicating that the stroke model was successfully established. Compared with the solvent group, the grid test failure rates in the treatment groups of target compounds 1, 5, and 11 were all significantly reduced, indicating that target compounds 1, 5, and 11 all have the efficacy of improving post-stroke motor dysfunction. Furthermore, compared with target compound 1, the grid test failure rates in PT mice after treatment with target compounds 5 and 11 were comparable and lower, indicating that target compounds 5 and 11 had a stronger effect on improving motor dysfunction in the mouse PT model than target compound 1.

[0069] The above results indicate that the 3-hydroxypyrazine-2-carboxylic acid dextranol or fumarate derivatives of the present invention can effectively improve stroke-induced motor dysfunction, and the target compounds 5 and 11 have stronger activity.

[0070] Example 5: Effect of target compounds 1, 5, 11 on improving cognitive impairment in 5×FAD mice.

[0071] The novel object recognition test is a learning and memory assessment method based on mice's innate tendency to explore new objects. The test consists of three phases: adaptation, learning, and testing. In the adaptation phase, mice are placed in an open field test chamber without any objects and allowed to explore freely for 5 minutes to adapt to the environment. In the learning phase, two identical objects are placed symmetrically in the open field, approximately 10 cm from the chamber walls. The mouse is then placed back-to-back in the open field test chamber and allowed to explore freely for 10 minutes. The testing phase occurs 24 hours later. One of the objects used in the learning phase is replaced with a new object of similar size but completely different in shape and color.

[0072] Five-fold FAD mice were placed in the experiment with their backs to the object, and the time spent exploring the old and new objects was recorded for 10 minutes. After each experiment, the experimental chamber was thoroughly wiped clean with 75% ethanol. Exploration behavior was defined as the mouse moving within 2 cm of the object while exhibiting sniffing behavior. Learning and memory were assessed by analyzing the exploration time of the mice for new or old objects and the discrimination index. The discrimination index was calculated as: (Time spent exploring the new object / Total time spent exploring both the new and old objects) × 100%.

[0073] The results are as follows Figure 3 As shown, compared with wild-type mice, 5×FAD mice exhibited a significantly lower discrimination index in the new object recognition test, indicating that 5×FAD mice are an ideal animal model for Alzheimer's disease. Compared with the solvent group, the discrimination indices in the new object recognition test were significantly increased in the treatment groups of target compounds 1, 5, and 11, indicating that target compounds 1, 5, and 11 all have the efficacy of improving cognitive dysfunction in Alzheimer's disease model animals. Furthermore, compared with target compound 1, the discrimination indices in the new object recognition test of 5×FAD mice were comparable and higher after treatment with target compounds 5 and 11, indicating that target compounds 5 and 11 have a stronger effect on improving cognitive dysfunction in Alzheimer's disease model animals than target compound 1.

[0074] The above results indicate that the 3-hydroxypyrazine-2-carboxylic acid dextranol or fumarate derivatives of the present invention can effectively improve cognitive impairment related to Alzheimer's disease, and the target compounds 5 and 11 have stronger activity.

Claims

1. A class of 3-hydroxypyrazine-2-carboxylic acid dextranol or frankinc ester derivatives thereof, characterized in that, Its chemical structure is shown in formula (I): Where: R 1 R 2 Each is independently an -H or C1-C6 alkyl group, R 3 for 2. The 3-hydroxypyrazine-2-carboxylic acid dextranol or frankincide derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, R 1 and R 2 Each is an alkyl group consisting of -H or C1-C3; preferably, R 1 and R 2 Each is independently -H or methyl; preferably, R 1 and R 2 Alkyl groups selected from C1-C3, more preferably, R 1 and R 2 It is -CH3.

3. The 3-hydroxypyrazine-2-carboxylic acid dextranol or frankincide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, characterized in that, R 3 for 4. The 3-hydroxypyrazine-2-carboxylic acid dextranol or frankincide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, characterized in that, R 3 for 5. The 3-hydroxypyrazine-2-carboxylic acid dextranol or frankincide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, characterized in that, R 4 For H.

6. The 3-hydroxypyrazine-2-carboxylic acid dextranol or frankincide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, characterized in that, R 4 for 7. The 3-hydroxypyrazine-2-carboxylic acid dextranol or fumarate derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, characterized in that, R 4 for 8.3-Hydroxypyrazine-2-carboxylic acid dextranol or fumarate derivatives thereof, or pharmaceutically acceptable salts thereof, characterized in that, The derivative is selected from any one or more of the following compounds:

9. A pharmaceutical composition comprising dextranol or fentanyl ester derivative of 3-hydroxypyrazine-2-carboxylic acid as described in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

10. The use of the 3-hydroxypyrazine-2-carboxylic acid dextranol or fumarate derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 8, or the composition of claim 9, in the preparation of a medicament for inhibiting an inflammatory response; preferably, in the preparation of a medicament for neuroinflammatory-related diseases; preferably, the neuroinflammatory-related diseases are selected from stroke, cerebral ischemia-reperfusion injury, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis.

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