Isoquinoline derivative, synthesis method therefor, and use thereof

Abstract

The present invention relates to the technical field of biomedicine, and disclosed are an isoquinoline derivative, a synthesis method therefor, and a use thereof. The isoquinoline derivative is a novel Pgk1 activator, has relatively high Pgk1 activation activity, has a better effect than terazosin used in the field at present, does not have the condition of inhibiting Pgk1, and has the potential of preventing or treating neurodegenerative diseases and metabolic diseases clinically.

Description

Isoquinoline derivative, synthesis method and application thereof Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an isoquinoline derivative, a synthesis method and an application thereof. Background Art

[0002] Reduced glucose metabolism in brain tissue is an important pathogenic factor in the occurrence of various neurodegenerative diseases.

[0003] The brain consumes 20-25% of the body's glucose, yet it only accounts for approximately 2% of body weight. This demonstrates the brain's enormous energy demands and its highly active glucose metabolism. However, glucose metabolism significantly decreases in the elderly brain. Numerous studies have shown that decreased brain glucose metabolism precedes the onset of clinical symptoms in Alzheimer's disease (AD) by 10-15 years. Decreased brain glucose metabolism is a common pathological feature of multiple neurodegenerative diseases. Why is glucose metabolism so crucial to the brain? First, glucose metabolism is the primary pathway for cellular energy (ATP). Neurons, as the most active cell type in the body, require large quantities of ATP to sustain various physiological activities, such as protein synthesis, long-distance transport of proteins and organelles, action potential generation, neurotransmitter release, and pinocytosis and exocytosis. Second, ATP prevents nonspecific protein aggregation, acting as a "hydrotrope." In the elderly, reduced glucose metabolism leads to a significant decrease in neuronal ATP concentration, potentially as low as 1-2 mM. In contrast, ATP levels in healthy individuals range from 8-10 mM. Low ATP concentrations lead to pathological aggregation of various proteins, while high ATP concentrations prevent nonspecific protein aggregation. Therefore, reduced glucose metabolism is a key factor in brain aging. Improving glucose metabolism has the potential to improve brain health and prevent, alleviate, or even reverse brain aging. However, there are no internationally recognized clinical drugs that can enhance glucose metabolism. Studies have found that resveratrol, metformin, rapamycin, vitamin B3, coenzyme Q10, creatine, and insulin have only ineffective or limited clinical effects. Currently, exercise and diet are widely recognized methods for protecting the brain and muscles. The beneficial effects of exercise and diet are primarily due to improved mitochondrial function and increased glucose metabolism in various organs.

[0004] Reduced glucose metabolism in muscle tissue is an important factor in the high incidence of insulin resistance in the elderly.

[0005] The global prevalence of diabetes among adults is approximately 10%. Diabetes is a disease associated with aging. One study showed that the prevalence of diabetes in people aged 20-39, 47.0%, 66.7%, and 75.7% in those aged 40-59, 60-74, and 75.7% respectively. Why are older adults more susceptible to diabetes? It is well known that over 80% of blood glucose is consumed by skeletal muscle. However, total muscle mass decreases by 8% per decade after age 40 and by 15% per decade after age 70, leading to a widespread prevalence of sarcopenia in the elderly. Even with normal insulin levels, individuals with sarcopenia are unable to metabolize blood glucose through skeletal muscle, leading to insulin resistance. Studies have found that for every 10% increase in skeletal muscle mass, the risk of insulin resistance decreases by 11%. In addition to sarcopenia, impaired glucose metabolism can be mediated by mitochondrial damage. Mitochondrial damage can be caused by a variety of factors, including fatty acid accumulation, oxidative stress, genetic mutations, and mitochondrial respiratory inhibitors, all of which increase the risk of type 2 diabetes.

[0006] In fact, impaired glucose metabolism often occurs simultaneously in multiple organs, including the brain and muscle tissue. Consequently, neurodegenerative diseases and insulin resistance often coexist in the same patient. Studies have found that 80% of patients with Alzheimer's disease and 58% of patients with Parkinson's disease have concomitant diabetes or insulin resistance.

[0007] Zozosins can prevent and treat neurodegenerative diseases by targeting Pgk1 and activating glucose metabolism.

[0008] Quinazolines are a class of α1-adrenergic receptor inhibitors used to treat benign prostatic hyperplasia (BPH) and hypertension. Common quinazolines on the market include terazosin (TZ), alfuzosin, and doxazosin. Previous research by our team has shown that terazosin binds to phosphoglycerate kinase 1 (Pgk1). The glycolysis pathway comprises ten enzymes, with Pgk1 being the seventh and the first to produce ATP. Terazosin activates Pgk1, thereby activating the glycolysis pathway. Pyruvate produced by the glycolysis pathway enters the mitochondrial tricarboxylic acid (TCA) cycle, increasing mitochondrial oxidative phosphorylation and generating large amounts of ATP. The glycolysis pathway and the mitochondrial tricarboxylic acid cycle cascade constitute the glucose metabolism pathway.

[0009] Studies have shown that terazosin has preventive and therapeutic efficacy in mouse and rat models of Parkinson's disease, Alzheimer's disease, and multiple system atrophy. This efficacy is supported by patient data. Multiple epidemiological analyses have shown that long-term use of terazosin-like drugs significantly reduces the incidence of Parkinson's disease and Parkinson's-related disorders. Other α1-adrenergic receptor inhibitors, such as tamsulosin, do not exhibit similar efficacy.

[0010] There are potential safety risks in zosin-type drugs, and the modification of zosin-type drugs is of research significance.

[0011] In principle, a drug that activates glucose metabolism should have a blood sugar-lowering effect. Results from multiple clinical trials have shown that terazosin or doxazosin can indeed improve blood sugar and lipid levels in diabetic patients, but the efficacy is extremely limited or ineffective. Furthermore, there are no reports of oxazolidinones lowering blood sugar in mice or rats. The combined use of an α1-adrenergic receptor inhibitor (terazosin) and a β-adrenergic receptor inhibitor (propranolol) did not affect blood sugar in rats. These results suggest that oxazolidinones are weakly or ineffective in activating glucose metabolism in skeletal muscle.

[0012] Adrenergic receptor inhibitors such as terazosin, alfuzosin, doxazosin, and tamsulosin can cause a variety of side effects, including intraoperative floppy iris syndrome (IFIS), hypotension, fatigue, dizziness, syncope, and blurred vision, leading to discontinuation of medication in 10-20% of patients. Studies have found that terazosin's binding to the α1-adrenergic receptor depends on a nitrogen atom on the quinazoline ring. Replacing this nitrogen atom with a carbon atom reduces its affinity for the α1-adrenergic receptor by over 1000-fold. Current research shows that the modified terazosin (TZ-md) still activates Pgk1 but does not inhibit the α1-adrenergic receptor.

[0013] Furthermore, low concentrations of terazosin can activate Pgk1, while high concentrations can inhibit it. This phenomenon is explained by the fact that the site where terazosin binds to the Pgk1 protein overlaps with the substrate ADP and the product ATP. Because terazosin has a binding affinity for Pgk1 that is over 10-fold higher than that for ADP, high doses of terazosin bind more to the Pgk1 protein, inhibiting the binding of the substrate ADP and, consequently, inhibiting Pgk1 enzyme activity. The rate-limiting step in the Pgk1 enzyme reaction is the dissociation of ATP from the Pgk1 protein, a process that accounts for nearly 96% of the enzyme reaction time. Terazosin has a binding affinity for Pgk1 that is over 100-fold higher than that for ATP. Therefore, low doses of terazosin can compete for ATP binding, accelerating the Pgk1 enzyme reaction and activating Pgk1. A recent prospective clinical trial found that some Parkinson's disease patients treated with terazosin experienced decreased ATP levels in brain tissue and red blood cells, unlike the elevated ATP levels seen in most patients. This result suggests that terazosin may have an inhibitory effect on Pgk1 in these patients, a possibility that is more likely to occur in patients with a disrupted blood-brain barrier. At the same time, existing studies have shown that 13.5% of patients with an average age of 70 have a disrupted blood-brain barrier.

[0014] In summary, zozosins, as Pgk1 activators, have three weaknesses: 1) They are inadequate in activating skeletal muscle glucose metabolism and lack hypoglycemic efficacy. Perhaps zozosins are not optimal compounds for Pgk1 activation. 2) Zozosins' inhibition of α1-adrenergic receptors has multiple adverse side effects, leading some patients to discontinue treatment. 3) Zozosins carry the risk of Pgk1 inhibition, potentially rendering them ineffective in treating related diseases.

[0015] Summary of the Invention

[0016] Based on the three shortcomings of the aforementioned oxazinoids, the present invention provides an isoquinoline derivative, its synthesis method, and its application. As a novel class of Pgk1 activators, the isoquinoline derivative exhibits high Pgk1 activation activity and has the potential to clinically prevent or treat neurodegenerative and metabolic diseases.

[0017] To achieve the above object, the present invention provides the following technical solutions:

[0018] One of the technical solutions of the present invention is to provide an isoquinoline derivative, the general structural formula of the isoquinoline derivative is as follows:

[0019] In the above formula, ring A represents an optionally substituted five-membered oxygen-containing heterocycle; X is O or C, Y is O or C, and X and Y contain at least one O; R 1 represents a hydrogen atom or a methyl group; R 2represents a hydrogen atom or a methyl group; R 3 is a five-membered cycloalkyl group or a five-membered heterocyclic group containing one heteroatom, wherein the heteroatom is selected from an oxygen atom and a nitrogen atom;

[0020] The structure of the isoquinoline derivatives is as follows:

[0021] The second technical solution of the present invention is to provide a pharmaceutically acceptable salt based on the above-mentioned isoquinoline derivatives.

[0022] Preferably, the pharmaceutically acceptable salt of the isoquinoline derivative includes hydrochloride, sulfate, hydrobromide, nitrate, trifluoroacetate, acetate, phosphate, benzenesulfonate, p-toluenesulfonate, methanesulfonate, oxalate, tartrate, fumarate, maleate, succinate, malate, benzoic acid, citrate or lactate.

[0023] The present invention verifies that hydrochloride, sulfate, and trifluoroacetate all have the same efficacy as isoquinoline derivatives, and other pharmaceutically acceptable salts should also have the same efficacy as the isoquinoline derivatives.

[0024] The third technical solution of the present invention: provides an isoquinoline derivative preparation, the active ingredient of which is the above-mentioned isoquinoline derivative and / or the pharmaceutically acceptable salt of the above-mentioned isoquinoline derivative.

[0025] Preferably, the preparation is various types of pharmaceutical preparations prepared by combining the above-mentioned isoquinoline derivatives or pharmaceutically acceptable salts of the above-mentioned isoquinoline derivatives with pharmaceutically acceptable excipients, such as water, alcohol, vegetable oil, starch, microcrystalline cellulose, lactose, gelatin, magnesium stearate, sodium alginate, maltodextrin, talc, cross-linked polyvinylpyrrolidone, gum and vaseline.

[0026] The fourth technical solution of the present invention: provides a phosphoglycerate kinase activator, which is: the above-mentioned isoquinoline derivative, the pharmaceutically acceptable salt of the above-mentioned isoquinoline derivative or the above-mentioned isoquinoline derivative preparation.

[0027] The fifth technical solution of the present invention: provides a drug for treating or preventing neurodegenerative diseases, wherein the drug is: the above-mentioned isoquinoline derivative, the above-mentioned pharmaceutically acceptable salt of the isoquinoline derivative or the above-mentioned isoquinoline derivative preparation.

[0028] Preferably, the neurodegenerative disease includes but is not limited to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis or chorea.

[0029] Technical solution six of the present invention: Provided is a drug for treating or preventing metabolic diseases, wherein the drug is: the above-mentioned isoquinoline derivative, the above-mentioned pharmaceutically acceptable salt of the isoquinoline derivative or the above-mentioned isoquinoline derivative preparation.

[0030] Preferably, the metabolic disease includes but is not limited to diabetes or obesity.

[0031] Technical solution seven of the present invention: provides a method for preparing the above-mentioned isoquinoline derivatives, including the following synthetic route:

[0032] Synthesis Route A:

[0033] Malonic acid and piperidine are added to a solution of compound I to obtain compound II after reaction; compound II reacts under a catalyst and H2 atmosphere to obtain compound III; compound III first reacts with oxalyl chloride to generate acyl chloride, and then obtains compound IV under the catalysis of aluminum chloride; compound IV reacts with isoamyl nitrite under acidic conditions to obtain compound V; compound V reacts with phosphorus oxychloride to obtain compound VI; compound VI reacts with an amino protecting agent in N-methylpyrrolidone to obtain compound VII; compound VII, compound XI, dioxane, Pd2(dba)3, RuPhos and cesium carbonate react together to obtain compound VIII; compound VIII removes the Boc protecting group to obtain compound IX; compound IX reacts with R 3 -COOH reaction to prepare compound X; compound X reacts with trifluoroacetic acid to remove amino protection to prepare the isoquinoline derivative compound T; the reaction scheme is as follows:

[0034] Preferably, the reaction temperature and reaction time of each step in the synthetic route A are not particularly limited as long as the raw materials of the reactants can produce the target product;

[0035] Synthesis route B:

[0036] Compound XI reacts with R 3 -COOH, O-(7-nitrobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine are reacted to prepare compound XII; compound XII is deprotected by Boc to obtain compound XIII; compound XIII is reacted with compound VII prepared according to synthesis route A, dioxane, Pd2(dba)3, RuPhos and cesium carbonate to obtain compound X; compound X is reacted with trifluoroacetic acid to remove amino protection to obtain the isoquinoline derivative compound T; the reaction route is as follows:

[0037] Preferably, the reaction temperature and reaction time of each step in the synthetic route B are not particularly limited as long as the raw materials of the reactants can produce the target product;

[0038] Synthesis Route C:

[0039] Compound VI prepared according to synthetic route A is reacted with ammonia to obtain compound XIV; compound XIV is reacted with an amino protecting agent to obtain compound VII; compound VII is used to prepare the isoquinoline derivative compound T according to the method of synthetic route A; the reaction scheme is as follows:

[0040] Preferably, the reaction temperature and reaction time of each step in Synthesis Route C are not particularly limited as long as the raw materials of the reactants can produce the target product.

[0041] The R of the compound involved in the synthetic route A, synthetic route B and synthetic route C 4 The group is an amino protecting group.

[0042] The beneficial technical effects of the present invention are as follows:

[0043] The synthesized isoquinoline derivatives and their salts have been shown to target and activate Pgk1, thereby treating or preventing neurodegenerative and metabolic diseases. They are more effective than terazosin and do not inhibit Pgk1. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG1 shows the effects of various isoquinoline derivatives and terazosin prepared in the examples of the present invention on cellular ATP activity.

[0045] Figure 2 shows the results of in vitro biochemical experiments on the effects of drugs on Pgk1 enzyme activity, where A is the positive control, showing the effects of different concentrations of terazosin on Pgk1 enzyme activity; and B is the experimental group, showing the effects of different concentrations of T6 on Pgk1 enzyme activity.

[0046] Figure 3 shows the effects of different concentrations of compound T6 and terazosin on the enzyme activity of Pgk1 in cells, wherein A is the effect of terazosin on the enzyme activity of Pgk1 in cells, and B is the effect of compound T6 on the enzyme activity of Pgk1 in cells.

[0047] FIG4 shows the effects of different concentrations of compound T6 and terazosin on ATP levels in cells.

[0048] FIG5 shows the effects of various isoquinoline derivatives and terazosin prepared in the examples of the present invention on blood glucose in mice.

[0049] Figure 6 shows the effects of liraglutide and different concentrations of compound T6 on blood glucose in diabetic model mice.

[0050] Figure 7 shows the effects of metformin, terazosin and compound T6 on the glycated hemoglobin, insulin content and body weight in the blood of diabetic model mice, where A is the glycated hemoglobin content, B is the insulin content, and C is the change in body weight.

[0051] Figure 8 shows the effects of terazosin and different concentrations of compound T2 on the motor ability and tyrosine hydroxylase content in MPTP-induced Parkinson's disease model mice, wherein A is the effect of each drug on the motor ability of MPTP-induced Parkinson's disease model mice, B is the effect of terazosin on the tyrosine hydroxylase content in MPTP-induced Parkinson's disease model mice, and C is the effect of 0.1 mg / Kg of compound T2 on the tyrosine hydroxylase content in MPTP-induced Parkinson's disease model mice. DETAILED DESCRIPTION

[0052] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0053] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intervening value in the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0055] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0056] Example 1: Synthesis of Compound T1-II

[0057] 35 mL of DMF was added to a 250 mL eggplant-shaped flask. With stirring, 9.01 g (60 mmol) of piperonal and 8.74 g (84.0 mmol) of malonic acid were added, followed by 0.66 g (7.8 mmol) of piperidine. The resulting light brown solution was stirred in a 90°C oil bath for 10 h. The solution was cooled to room temperature, 60 mL of purified water was added, and stirred for 1 h. The mixture was filtered, and the filter cake was washed with purified water and dried under vacuum at room temperature to yield 8.67 g of light brown intermediate T1-II (yield: 75.2%). NMR analysis is as follows:

[0058] 1 H NMR: (300MHz, DMSO-d6) δ12.24(brs,1H),7.52(d,J=15.9Hz,1H),7.37(d,J=1.6Hz,1H),7.1 6(dd,J1=8.1Hz,J2=1.6Hz,1H),6.95(d,J=8.1Hz,1H),6.40(d,J=15.9Hz,1H),6.08(s,2H).

[0059] MS(C 10 H8O4):191.67[MH] - ,383.85[2M-H] - .

[0060] Example 2: Synthesis of Compound T1-III

[0061] 250 mL of tetrahydrofuran was added to a 500 mL eggplant-shaped flask, and 4.19 g (2.18 mmol) of intermediate T1-II and 0.84 g of palladium on carbon were added with stirring. The resulting mixture was stirred at room temperature under a hydrogen atmosphere for 20 h. The reaction mixture was filtered through celite, the filtrate was concentrated under reduced pressure, and the residue was recrystallized from ethyl acetate / petroleum ether to obtain 2.97 g of white intermediate T1-III, with a yield of 70.1%. NMR analysis is as follows:

[0062] 1 H NMR: (300MHz, CDCl3) δ11.81(brs,1H),6.74(d,J=7.9Hz,1H),6.70(d,J=1.4Hz,1H),6.66 (dd, J1=7.9Hz, J2=1.7Hz, 1H), 5.93 (s, 2H), 2.88 (t, J=7.8Hz, 2H), 2.64 (t, J=7.8Hz, 2H).

[0063] MS(C 10 H 10 O4):193.57[MH] - ,387.79[2M-H]- .

[0064] Example 3: Synthesis of Compound T1-IV

[0065] 50 mL of dichloromethane was added to a 250 mL eggplant-shaped flask, and 4.40 g (22.66 mmol) of intermediate T1-III and 0.17 mL of DMF were added with stirring. The resulting light brown solution was stirred in an ice bath. 5.75 g (45.32 mmol) of oxalyl chloride was added dropwise to the reaction solution with stirring. The resulting reaction solution was gradually warmed to room temperature and stirred overnight (approximately 12 h). The reaction solution was concentrated by rotary evaporation under vacuum to obtain a brown-yellow oil. 80 mL of dichloromethane was added again to the ice bath, and 4.53 g (33.99 mmol) of aluminum chloride (added in three equal portions) was added. The resulting reddish-brown mixture was stirred in the ice bath for 3 h. 80 g of ice water (added in three equal portions) was added, and the mixture was stirred for 0.5 h. The mixture was separated, and the aqueous phase was extracted twice with dichloromethane (40 mL) (the volumes in parentheses represent the amount of solution used for each extraction or washing, and have the same meaning in the following examples). The combined organic phases were washed three times with saturated sodium bicarbonate (100 mL) and once with saturated sodium chloride (50 mL), dried over anhydrous sodium sulfate, and the solvent removed by rotary evaporation in vacuo. The resulting residue was purified by column chromatography (dichloromethane:methanol = 200:1-150:1-100:1) to obtain 2.37 g of intermediate T1-IV, yield: 59.4%. NMR analysis is as follows:

[0066] 1 H NMR: (300MHz, CDCl3) δ7.03(s,1H),6.79(s,1H),6.02(s,2H),2.97(m,2H),2.62(m,2H).

[0067] MS(C 10 H8O3):176.96[M+H] + .

[0068] Example 4: Synthesis of Compound T1-V

[0069] 65 mL of methanol was added to a 100 mL eggplant-shaped flask. 6.52 g (37.01 mmol) of intermediate T1-IV and 1.08 mL (12.95 mmol) of concentrated hydrochloric acid were added with stirring. 6.50 g (55.51 mmol) of isoamyl nitrite was added dropwise to the resulting nearly colorless solution and stirred at room temperature for 8 h. The resulting reaction mixture was filtered, and the filter cake was washed with methanol (15 mL) and dried under vacuum at room temperature to yield 7.43 g of yellow intermediate T1-V (yield: 97.8%). NMR analysis is as follows:

[0070] 1 H NMR: (300MHz, DMSO-d6) δ12.44(s,1H),7.11(s,1H),7.09(s,1H),6.17(s,2H),3.62(s,2H).

[0071] MS(C 10 H7NO4):223.11[M+H2O] + ,411.17[2M+H] + ,433.11[2M+Na] + .

[0072] Example 5: Synthesis of Compound T1-VI

[0073] 7.43 g of intermediate T1-V was added to a 250 mL eggplant-shaped flask, along with 75 mL of phosphorus oxychloride and 0.75 mL of DMF. The resulting yellow mixture was stirred in an oil bath at 100°C for 4 hours. After completion of the reaction, the phosphorus oxychloride was removed by rotary evaporation in vacuo. The residue was purified by column chromatography (dichloromethane:methanol = 400:1) to afford 6.47 g of intermediate T1-VI in a yield of 73.8%. NMR analysis is as follows:

[0074] 1 H NMR: (300MHz, DMSO-d6) δ7.86(s,1H),7.48(s,1H),7.41(s,1H),6.30(s,2H).

[0075] MS(C 10 H5Cl2NO2):241.99[M+H] + .

[0076] Example 6: Synthesis of Compound T1-VII

[0077] 4.63 g (19.12 mmol) of intermediate T1-VI, 7.87 g (57.35 mmol) of p-methoxybenzylamine and 46 mL of N-methylpyrrolidone were added to a 250 mL reaction tube, and the resulting reaction mixture was reacted in a 120°C oil bath under nitrogen for 3 h. After the reaction, 150 mL of water and 150 mL of ethyl acetate were added, and the organic phase was collected. The aqueous phase was extracted twice with ethyl acetate (50 mL), and the combined organic phases were washed once with saturated sodium chloride (50 mL) and dried over anhydrous sodium sulfate. The solvent was removed by vacuum rotary evaporation, and the resulting residue was purified by column chromatography (ethyl acetate: petroleum ether = 1:20-1:10-1:5-1:3-1:2) to obtain 5.51 g of intermediate T1-VII, with a yield of 84.0%. NMR analysis is as follows:

[0078] 1 H NMR: (300MHz, DMSO-d6)δ7.87(t,J=5.7Hz,1H),7.73(s,1H),7.31(brs,1H),7.28(brs,1H),7.1 1(s,1H),6.89-6.88(m,1H),6.86-6.85(m,2H),6.15(s,2H),4.58(d,J=5.7Hz,2H),3.71(s,3H).

[0079] MS(C 18 H 15 ClN2O3):343.09[M+H] + .

[0080] Example 7: Synthesis of Compound T1-VIII

[0081] To a 200 mL reaction tube was added dioxane (50 mL), 3.43 g (10.00 mmol) of intermediate T1-VII, 3.73 g (20.00 mmol) of N-Boc-piperazine, 30.73 g (0.80 mmol) of Pd2(dba), 0.56 g (1.2 mmol) of RuPhos, and 6.52 g (20.00 mmol) of cesium carbonate. The resulting reaction mixture was stirred at 100°C under nitrogen for 12 h. After the reaction was completed, the mixture was cooled to room temperature. The reaction mixture was filtered through celite, and 150 mL of water and 150 mL of ethyl acetate were added to the filtrate. The organic phase was collected and the aqueous phase was extracted twice with ethyl acetate (50 mL). The combined organic phases were washed once with saturated sodium chloride (50 mL) and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation in vacuo, and the resulting residue was purified by column chromatography (ethyl acetate:petroleum ether = 1:40-1:20-1:10-1:5-1:3) to obtain 3.46 g of intermediate T1-VIII, yield: 70.2%. NMR analysis is as follows:

[0082] 1 H NMR: (300MHz, CDCl3) δ7.35-7.33(m,1H),7.32-7.30(m,1H),6.90-6.89(m,2H),6.87-6.85(m,1H),6.81(s,1H),6.06 (s,1H),5.96(s,2H),4.98(m,1H),4.68-4.66(m,2H),3.81(s,3H),3.58-3.53(m,4H),3.46-3.43(m,4H),1.49(s,9H).

[0083] MS(C 27 H 32N4O5):493.28[M+H] + .

[0084] Example 8: Synthesis of Compound T1-IX

[0085] To a 100 mL eggplant flask, 1.38 g (2.80 mmol) of intermediate T1-VIII and 27 mL of 4N hydrochloric acid / ethyl acetate solution were added. The resulting reaction mixture was stirred at room temperature for 7 h. The solvent was removed by rotary evaporation in vacuo. The resulting residue was washed with ethyl acetate (10 mL), filtered, and dried in vacuo at room temperature to yield 1.10 g of intermediate T1-IX. The product was carried on to the next step without further purification.

[0086] MS(C 22 H 25 ClN4O3):393.32[M+H-HCl] + .

[0087] Example 9: Synthesis of Compound T1-X

[0088] To a 50 mL eggplant-shaped flask were added 536 mg (1.25 mmol) of intermediate T1-IX, 175 mg (1.5 mmol) of (R)-(+)-tetrahydrofuran-2-carboxylic acid, 713 mg (1.875 mmol) of HATU, 485 mg (3.75 mmol) of DIEA, and 10 mL of DMF. The resulting reaction mixture was stirred at room temperature overnight under nitrogen. Water (100 mL) and ethyl acetate (100 mL) were added, and the organic phase was collected. The aqueous phase was extracted twice with ethyl acetate (30 mL). The combined organic phases were washed once with saturated sodium chloride (30 mL) and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (ethyl acetate:petroleum ether = 1:20-1:10-1:5-1:3-1:1-2:1-3:1) to obtain 324 mg of intermediate T1-X in a yield of 52.9%. NMR analysis is as follows:

[0089] 1 H NMR: (300MHz, CDCl3)δ7.34-7.33(m,1H),7.31(m 1H),6.89-6.85(m,3H),6.82(s,1H),6.06(s,1H),5.97(s,2H),5.01(brs,1H),4.67(m,3H),3.98-3. 86(m,3H),3.81(s,3H),3.76-3.67(m,3H),3.53-3.47(m,4H),2.32-2.28(m,1H),2.08-1.92(m,3H).

[0090] MS(C 27 H 30 N4O5):491.28[M+H] + .

[0091] Example 10: Synthesis of Compound T1

[0092] 200 mg (0.41 mmol) of intermediate T1-X and 2 mL of trifluoroacetic acid were added to a 4 mL reaction tube. The resulting reaction mixture was stirred in a 45 ° C oil bath for 3 h, and the solvent was removed by vacuum rotary evaporation. 15 mL of saturated NaHCO3 solution was added to the resulting residue, and the mixture was extracted three times with dichloromethane (15 mL). The organic phases were combined, washed once with saturated sodium chloride (15 mL), and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (dichloromethane: methanol = 100:1-80:1-50:1-20:1-10:1) to obtain 62 mg of compound T1, with a yield of 41.1%. NMR analysis is as follows:

[0093] 1 H NMR: (300MHz, DMSO-d6)δ7.60(s,1H),7.00(s,1H),6.55(brs,2H),6.25(s,1H),6.10(s,2H ),4.68(m,1H),3.81-3.70(m,4H),3.65-3.45(m,6H),2.09-1.98(m,2H),1.88-1.79(m,2H).

[0094] MS(C 19 H 22 N4O4):371.05[M+H] + .

[0095] Example 11: Synthesis of Compound T1-XII

[0096] 1.16 g (10.0 mmol) of (R)-(+)-tetrahydrofuran-2-carboxylic acid and 20 ml of DMF were added to a 100 ml reaction tube. To the resulting reaction solution were added 1.86 g (10.0 mmol) of intermediate T1-XI, 4.56 g (12.0 mmol) of HATU, and 2.59 g (20.0 mmol) of N,N-diisopropylethylamine. The resulting reaction solution was stirred at room temperature for 12 h. After the reaction was completed, 100 mL of water and 100 mL of ethyl acetate were added, and the organic phase was collected. The aqueous phase was extracted twice with ethyl acetate (50 mL). The combined organic phases were washed once with saturated sodium chloride (50 mL) and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation in vacuo, and the resulting residue was purified by column chromatography (ethyl acetate:petroleum ether = 1:20-1:10-1:7-1:5-1:2) to obtain 1.95 g of intermediate T1-XII, with a yield of 68.7%. NMR analysis is as follows:

[0097] 1 H NMR: (300MHz, DMSO-d6) δ4.56-4.52(m,1H),3.90-3.75(m,2H),3.63-3.59(m ,2H),3.44-3.28(m,6H),2.32-2.21(m,1H),1.99-1.87(m,3H),1.41(s,9H).

[0098] MS(C 14 H 24 N2O4):285.14[M+H] + .

[0099] Example 12: Synthesis of Compound T1-XIII

[0100] To a 100 mL eggplant flask, 1.42 g (5.0 mmol) of intermediate T1-XII and 28 mL of 4N hydrochloric acid / ethyl acetate solution were added. The resulting reaction mixture was stirred at room temperature for 5 h. The solvent was removed by rotary evaporation in vacuo. The resulting residue was washed with ethyl acetate (10 mL), filtered, and dried in vacuo at room temperature to yield 0.95 g of intermediate T1-XIII. The product was carried on to the next step without further purification.

[0101] MS(C9H 17 ClN2O2):185.17[M+H-HCl] + .

[0102] Example 13: Synthesis of Compound T1-X

[0103] To a 200 mL reaction tube was added dioxane (40 mL), 2.58 g (7.50 mmol) of intermediate T1-VII, 3.3 g (15.00 mmol) of intermediate T1-XIII, 0.55 g (0.60 mmol) of Pd2(dba)3, 0.42 g (0.90 mmol) of RuPhos, and 4.89 g (15.00 mmol) of cesium carbonate. The resulting reaction mixture was stirred at 100°C under nitrogen for 12 h. After the reaction was completed, the mixture was cooled to room temperature. The reaction mixture was filtered through celite, and 120 mL of water and 120 mL of ethyl acetate were added to the filtrate. The organic phase was collected and the aqueous phase was extracted twice with ethyl acetate (50 mL). The combined organic phases were washed once with saturated sodium chloride (50 mL) and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation in vacuo, and the resulting residue was purified by column chromatography (ethyl acetate:petroleum ether=1:20-1:10-1:5-1:3-1:1-2:1-3:1) to give 1.85 g of intermediate T1-X in a yield of 50.1%.

[0104] Example 14: Synthesis of Compounds T1-XIV

[0105] To a 100mL hydrothermal reactor, 1.09g (4.50mmol) of intermediate T1-VI, 30mL of dioxane and 30mL of ammonia were added, and the resulting reaction mixture was stirred at 150°C for 48h. After the reaction was completed, it was cooled to room temperature. 30mL of water and 30mL of dichloromethane were added to the reaction mixture, the organic phase was collected, the aqueous phase was extracted twice with dichloromethane (15mL), and the combined organic phases were washed once with saturated sodium chloride (30mL) and dried over anhydrous sodium sulfate. The solvent was removed by vacuum rotary evaporation, and the resulting residue was pulped twice with ethyl acetate (20mL) at room temperature to give 0.64g of intermediate T1-XIV, a yield of 64.0%. 1 H NMR analysis is as follows:

[0106] 1 H NMR: (300MHz, DMSO-d6) δ7.61(s,1H),7.10(s,1H),6.94(brs,2H),6.84(s,1H),6.14(s,2H).

[0107] MS(C 10 H7ClN2O2):223.08[M+H] + .

[0108] Example 15: Synthesis of Compound T1-VII

[0109] To a 100 mL hydrothermal reactor were added 520 mg (2.34 mmol) of intermediate T1-XIV, 79 mg (0.35 mmol) of palladium acetate, 256 mg (0.70 mmol) of sodium 3-diphenylphosphinobenzenesulfonate, 1.29 g (9.36 mmol) of p-methoxybenzyl alcohol, and 15 mL of water. The resulting reaction mixture was stirred at 140°C for 24 h. After completion of the reaction, the reaction mixture was filtered through celite, 30 mL of water and 30 mL of ethyl acetate were added, and the organic phase was collected. The aqueous phase was extracted twice with ethyl acetate (15 mL). The combined organic phases were washed once with saturated sodium chloride (30 mL) and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation in vacuo, and the resulting residue was purified by column chromatography (ethyl acetate:petroleum ether = 1:20-1:10-1:7-1:5-1:2) to obtain 592 mg of intermediate T1-VII in a yield of 74.0%.

[0110] Example 16: Synthesis of Compound T2

[0111] The synthesis of starting material T2-X refers to the synthesis method of T1-X.

[0112] 180 mg (0.37 mmol) of intermediate T2-X and 1.8 mL of trifluoroacetic acid were added to a 4 mL reaction tube. The resulting reaction mixture was stirred in a 45 ° C oil bath for 3 h, and the solvent was removed by vacuum rotary evaporation. 15 mL of saturated NaHCO3 solution was added to the resulting residue, and the mixture was extracted three times with dichloromethane (15 mL). The organic phases were combined, washed once with saturated sodium chloride (15 mL), and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (dichloromethane: methanol = 100:1-80:1-50:1-20:1-10:1) to give 65 mg of compound T2, with a yield of 47.8%. NMR analysis is as follows:

[0113] 1 H NMR: (300MHz, DMSO-d6) δ7.46(s,1H),6.89(s,1H),6.44(brs,2H),6.10(s,1H),6.04(s,2H ),4.71(m,1H),3.82-3.70(m,4H),3.66-3.56(m,6H),2.07-1.99(m,2H),1.88-1.80(m,2H).

[0114] MS(C 19 H 22 N4O4):371.20[M+H] + .

[0115] Example 17: Synthesis of Compound T3

[0116] The synthesis of starting material T3-X refers to the synthesis method of T1-X.

[0117] 200 mg (0.37 mmol) of intermediate T3-X and 2.0 mL of trifluoroacetic acid were added to a 4 mL reaction tube. The resulting reaction mixture was stirred in a 45°C oil bath for 3 h, and the solvent was removed by vacuum rotary evaporation. 15 mL of saturated NaHCO3 solution was added to the resulting residue, and the mixture was extracted three times with dichloromethane (15 mL). The organic phases were combined, washed once with saturated sodium chloride (15 mL), and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (dichloromethane:methanol = 100:1-80:1-50:1-20:1-15:1) to give 78 mg of compound T3, with a yield of 51.3%. NMR analysis is as follows:

[0118] 1 H NMR: (300MHz, DMSO-d6) δ7.42(s,1H),6.86(s,1H),6.52(brs,2H),6.30(q,J=5.0Hz,1H),6.19(s,1H),4.75 (m,1H),3.85-3.68(m,4H),3.72-3.60(m,6H),2.05-1.98(m,2H),1.90-1.80(m,2H),1.67(d,J=5.0Hz,3H).

[0119] MS(C 20 H 24 N4O4):385.23[M+H] + .

[0120] Example 18: Synthesis of Compound T4

[0121] The synthesis of starting material T4-X refers to the synthesis method of T1-X.

[0122] 182 mg (0.35 mmol) of intermediate T4-X and 1.9 mL of trifluoroacetic acid were added to a 4 mL reaction tube. The resulting reaction mixture was stirred in a 45°C oil bath for 3 h, and the solvent was removed by vacuum rotary evaporation. 15 mL of saturated NaHCO3 solution was added to the resulting residue, and the mixture was extracted three times with dichloromethane (15 mL). The organic phases were combined, washed once with saturated sodium chloride (15 mL), and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (dichloromethane:methanol = 100:1-80:1-50:1-20:1-15:1) to give 78 mg of compound T4, with a yield of 55.7%. NMR analysis is as follows:

[0123] 1H NMR: (300MHz, DMSO-d6) δ7.53(s,1H),6.95(s,1H),6.56(brs,2H),6.15(s,1H),4.76(m,1H ),3.80-3.66(m,4H),3.70-3.55(m,6H),2.10-1.99(m,2H),1.86-1.78(m,2H),1.72(s,6H).

[0124] MS(C 21 H 26 N4O4):399.25[M+H] + .

[0125] Example 19: Synthesis of Compound T5

[0126] The synthesis of starting material T5-X refers to the synthesis method of T1-X.

[0127] 245 mg (0.50 mmol) of intermediate T5-X and 2.5 mL of trifluoroacetic acid were added to a 4 mL reaction tube. The resulting reaction mixture was stirred in a 45°C oil bath for 3 h, and the solvent was removed by vacuum rotary evaporation. 15 mL of saturated NaHCO3 solution was added to the resulting residue, and the mixture was extracted three times with dichloromethane (15 mL). The organic phases were combined, washed once with saturated sodium chloride (15 mL), and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (dichloromethane:methanol = 100:1-80:1-50:1-20:1-15:1) to give 85 mg of compound T5, with a yield of 45.9%. NMR analysis is as follows:

[0128] 1 H NMR: (300MHz, DMSO-d6) δ7.98(s,1H),7.17(s,1H),6.50(s,2H),6.29(s,1H),4.62(t,J=8.6Hz,2H),3.26( t,J=8.6Hz,2H),4.72(m,1H),3.84-3.71(m,4H),3.66-3.48(m,6H),2.10-2.00(m,2H),1.90-1.80(m,2H).

[0129] MS(C 20 H 24 N4O3):369.15[M+H] + .

[0130] Example 20: Synthesis of Compound T6

[0131] The synthesis of starting material T6-X refers to the synthesis method of T1-X.

[0132] 220 mg (0.45 mmol) of intermediate T6-X and 2.2 mL of trifluoroacetic acid were added to a 4 mL reaction tube. The resulting reaction mixture was stirred in a 45°C oil bath for 3 h, and the solvent was removed by vacuum rotary evaporation. 15 mL of saturated NaHCO3 solution was added to the resulting residue, and the mixture was extracted three times with dichloromethane (15 mL). The organic phases were combined, washed once with saturated sodium chloride (15 mL), and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (dichloromethane:methanol = 100:1-80:1-50:1-20:1-15:1) to obtain 80 mg of compound T6, with a yield of 48.2%. NMR analysis is as follows:

[0133] 1 H NMR: (300MHz, DMSO-d6) δ7.92(s,1H),7.23(s,1H),6.53(s,2H),6.25(s,1H),4.64(t,J=8.6Hz,2H),3.24( t,J=8.6Hz,2H),4.75(m,1H),3.88-3.70(m,4H),3.69-3.52(m,6H),2.11-2.00(m,2H),1.88-1.80(m,2H).

[0134] MS(C 20 H 24 N4O3):369.21[M+H] + .

[0135] Example 21: Synthesis of Compound T7

[0136] The synthesis of starting material T7-X refers to the synthesis method of T1-X.

[0137] 115 mg (0.24 mmol) of intermediate T7-X and 1.2 mL of trifluoroacetic acid were added to a 4 mL reaction tube. The resulting reaction mixture was stirred in a 45 ° C oil bath for 3 h, and the solvent was removed by vacuum rotary evaporation. 15 mL of saturated NaHCO3 solution was added to the resulting residue, and the mixture was extracted three times with dichloromethane (15 mL). The organic phases were combined, washed once with saturated sodium chloride (15 mL), and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (dichloromethane: methanol = 100:1-80:1-50:1-20:1) to give 34 mg of compound T7, yield: 39.2%. NMR analysis is as follows:

[0138] 1H NMR: (300MHz, DMSO-d6) δ7.58(s,1H),7.00(s,1H),6.82-6.76(m,2H),6.21(s,1H),6. 11(s,2H),3.61-3.60(m,4H),3.30-3.25(m,4H),3.08-2.98(m,1H),1.79-1.52(m,8H).

[0139] MS(C 20 H 24 N4O3):369.22[M+H] + .

[0140] Example 22: Synthesis of Compound T8

[0141] The synthesis of starting material T8-X refers to the synthesis method of T1-X.

[0142] 79 mg (0.13 mmol) of intermediate T8-X and 1.6 mL of trifluoroacetic acid were added to a 4 mL reaction tube, and the resulting reaction mixture was stirred in a 45°C oil bath for 3 h. The solvent was removed by vacuum rotary evaporation. 15 mL of saturated NaHCO3 solution was added to the resulting residue, and the mixture was extracted three times with dichloromethane (15 ml). The organic phases were combined, washed once with saturated sodium chloride (15 ml), and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (dichloromethane:methanol = 100:1-80:1-50:1-20:1-8:1) to give 34 mg of compound T8, with a yield of 68.7%. NMR analysis is as follows:

[0143] 1 H NMR: (300MHz, DMSO-d6) δ9.72(brs,1H),8.53(brs,1H),7.53(s,1H),6.96(s,1H),6.17(s,1H),6.0 8(s,2H),4.67(m,1H),3.71-3.61(m,4H),3.39-3.18(m,7H),2.44-2.36(m,1H),1.95-1.81(m,3H).

[0144] MS(C 19 H 23 N5O3):370.27[M+H] + .

[0145] Example 23: Synthesis of Compound T9

[0146] The synthesis of starting material T9-X refers to the synthesis method of T1-X.

[0147] 120 mg (0.20 mmol) of intermediate T9-X and 2.4 mL of trifluoroacetic acid were added to a 4 mL reaction tube, and the resulting reaction mixture was stirred in a 45°C oil bath for 3 h. The solvent was removed by vacuum rotary evaporation. 15 mL of saturated NaHCO3 solution was added to the resulting residue, and the mixture was extracted three times with dichloromethane (15 mL). The organic phases were combined, washed once with saturated sodium chloride (15 mL), and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (dichloromethane:methanol = 100:1-80:1-50:1-20:1-8:1) to give 55 mg of compound T9, with a yield of 73.3%. NMR analysis is as follows:

[0148] 1 H NMR: (300MHz, DMSO-d6) δ9.69(brs,1H),8.55(brs,1H),7.55(s,1H),6.88(s,1H),6.22(s,1H),6.1 1(s,2H),4.61(m,1H),3.80-3.65(m,4H),3.45-3.21(m,7H),2.52-2.33(m,1H),2.00-1.80(m,3H).

[0149] MS(C 19 H 23 N5O3):370.25[M+H] + .

[0150] Example 24: Synthesis of Compound T10

[0151] The synthesis of starting material T10-X refers to the synthesis method of T1-X.

[0152] 170 mg (0.34 mmol) of intermediate T10-X and 1.7 mL of trifluoroacetic acid were added to a 4 mL reaction tube and stirred in a 45 ° C oil bath for 3 h. The solvent was removed by vacuum rotary evaporation. 15 mL of saturated NaHCO3 solution was added to the resulting residue and extracted three times with dichloromethane (15 mL). The organic phases were combined, washed once with saturated sodium chloride (15 mL), and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure and the resulting residue was purified by column chromatography (dichloromethane: methanol = 100:1-80:1-50:1-20:1-10:1) to give 48 mg of compound T10, yield: 37.1%. NMR analysis is as follows:

[0153] 1H NMR: (300MHz, DMSO-d6) δ8.50(br,2H),7.91(s,1H),7.16(s,1H),6.33(s,1H),4.75-4.72(m,1H),4.42-4. 35(m,4H),3.83-3.76(m,2H),3.73-3.56(m,4H),3.26-3.10(m,4H),2.14-1.97(m,2H),1.90-1.79(m,2H).

[0154] MS(C 20 H 24 N4O4):385.05[M+H] + .

[0155] Example 25: Synthesis of Compound T11

[0156] The synthesis of starting material T11-X refers to the synthesis method of T1-X.

[0157] 77 mg (0.16 mmol) of intermediate T11-X and 1.5 mL of trifluoroacetic acid were added to a 4 mL reaction tube. The resulting reaction mixture was stirred in a 45°C oil bath for 3 h, and the solvent was removed by vacuum rotary evaporation. 15 mL of saturated NaHCO3 solution was added to the resulting residue, and the mixture was extracted three times with dichloromethane (15 mL). The organic phases were combined, washed once with saturated sodium chloride (15 mL), and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (dichloromethane:methanol = 100:1-80:1-50:1-20:1-10:1) to give 38 mg of compound T11, with a yield of 65.5%. NMR analysis is as follows:

[0158] 1 H NMR: (300MHz, DMSO-d6)δ7.87-7.86(m,1H),7.56(s,1H),7.21-7.18(m,1H),7.05-7.0 4(m,1H),6.97(s,1H),6.93-6.79(m,1H),6.65-6.64(m,1H),6.19(s,1H),6.10(s,2H), 3.80(br,4H),3.41-3.39(m,4H).

[0159] MS(C 19 H 18 N4O4):367.23[M+H] + .

[0160] Example 26: Preparation of Compound T1 Trifluoroacetate

[0161] 250 mg (0.51 mmol) of intermediate T1-X and 2.5 mL of trifluoroacetic acid were added to a 4 mL reaction tube. The resulting reaction mixture was stirred in a 45°C oil bath for 3 h, and the solvent was removed by rotary evaporation in vacuo. The resulting residue was washed twice with methanol (2.5 mL), filtered, and dried in vacuo at room temperature to yield 112 mg of compound T1 trifluoroacetate salt (yield: 45.3%). NMR analysis is as follows:

[0162] 1 H NMR: (300MHz, DMSO-d6) δ7.44(s,1H),6.88(s,1H),6.32(brs,2H),6.08(s,1H),6.03(s,2H ),4.71(m,1H),3.82-3.70(m,4H),3.66-3.55(m,6H),2.08-1.99(m,2H),1.88-1.80(m,2H).

[0163] MS(C 21 H 23 F3N4O6):371.14[M+H-TFA] + .

[0164] Example 27: Preparation of Compound T1 Hydrochloride

[0165] 120 mg of compound T1 and 2.4 mL of methanol were added to a 4 mL reaction tube. The resulting mixture was stirred and cooled in an ice-water bath. 0.054 mL of concentrated hydrochloric acid was added. The resulting mixture was stirred in an ice bath for 1 hour and then filtered. The filter cake was rinsed with icy methanol and dried under vacuum to obtain 103 mg of compound T1 hydrochloride, yield: 78.0%. NMR analysis is as follows:

[0166] 1 H NMR: (300MHz, DMSO-d6)δ8.78(brs,3H),7.48(s,1H),6.91(s,1H),6.11(s,1H),6.05(s,2H ),4.74(m,1H),3.84-3.71(m,4H),3.70-3.57(m,6H),2.10-2.01(m,2H),1.90-1.80(m,2H).

[0167] MS(C 19 H 23 ClN4O4):371.19[M+H-HCl] + .

[0168] Example 28: Preparation of Compound T1 Sulfate

[0169] 200 mg of compound T1 and 4.0 mL of methanol were added to a 4 mL reaction tube. The resulting mixture was stirred and cooled in an ice-water bath. 0.058 mL of concentrated sulfuric acid was added. The resulting mixture was stirred in an ice bath for 1 hour and then filtered. The filter cake was rinsed with icy methanol and dried under vacuum to yield 213 mg of compound T1 sulfate (yield: 84.2%). 1 H NMR analysis is as follows:

[0170] 1 H NMR: (300MHz, DMSO-d6)δ8.89(brs,2H),7.46(s,1H),6.90(s,1H),6.13(s,1H),6.06(s,2H ),4.75(m,1H),3.83-3.69(m,4H),3.68-3.55(m,6H),2.10-1.99(m,2H),1.86-1.78(m,2H).

[0171] MS(C 19 H 24 N4O8S):371.25[M+H-H2SO4] + .

[0172] Example 29: Determining the Effect of Compounds on Cellular Glycolysis

[0173] Effects of compounds on cellular ATP levels.

[0174] Experimental conditions:

[0175] SK-N-SH (Human Neuroblastoma Cell Line) is a human neuroblastoma cell line. SK-N-SH cells were seeded into 6 empty plates at a density of 6×10 4 . Incubate for 12 hours under 5% O2 conditions. Add 10 μM of various drugs. After 48 hours, collect the cells. Measure the cellular ATP level using the Promega ATP detection kit. Repeat the experiment 6-10 times. The experimental results are shown in Figure 1. The ATP level of the untreated group (control) is defined as 1, and the terazosin-treated group is the positive control. T1 to T11 are the experimental groups.

[0176] Experimental results: T1-T9 can increase cell ATP activity; while T10 and T11 do not increase ATP activity.

[0177] Example 30: Determining the affinity of compounds for Pgk1 protein

[0178] Experimental conditions:

[0179] Purified Pgk1 protein (1 mg / mL) was added to a Biacore T200 chip. After confirming protein coupling, different concentrations of drug were added. The resulting curves were used to calculate parameters such as affinity and Rmax.

[0180] The experimental results are shown in Table 1.

[0181] Table 1

[0182] Experimental Results: Table 1 lists the affinity of various drugs for Pgk1. Among them, terazosin has the highest affinity, reaching 1.4μM. T1-T9 all have affinity for the Pgk1 protein, but the affinity is much lower than that of terazosin. T10 and T11 have no detectable affinity for the Pgk1 protein (KD value greater than 1000μM). Rmax (RU) refers to the amount of small molecule drug bound to the target protein when affinity is detected. Our published studies have shown that terazosin interferes with ATP, promoting the dissociation of ATP from the Pgk1 protein, thereby relieving the inhibitory effect of ATP on the Pgk1 enzyme reaction. The higher the Rmax value, the greater the potential to interfere with ATP dissociation. ADP is the substrate of Pgk1, with a KD value of 337.4. When measuring terazosin affinity, the addition of saturated ADP reduced the KD value for terazosin from 1.4 μM to 2.9 μM, indicating that the binding site of terazosin on the Pgk1 protein overlaps with the ADP / ATP complex. The KD value of T6 was also significantly reduced after the addition of ADP. This suggests that the binding of T6 to Pgk1 can be disrupted by ADP, indicating that the binding site of T6 on the Pgk1 protein is similar to that of terazosin. This result reveals that several isoquinoline derivatives have affinity for Pgk1, but the affinity is weaker than that of terazosin.

[0183] Example 31: Determining the Effect of Compounds on Mitochondrial Respiration

[0184] Experimental conditions:

[0185] Mitochondrial stress was assessed using a Seahorse XFe analyzer (XFe-24, Seahorse Bioscience, Billerica, MA, UAS) and oxygen consumption rate (OCR). SH-SKN cells were seeded in 24-well plates compatible with the Seahorse XFe analyzer. After 24 hours, cells were treated with DMSO (0.01%) or various drugs (10 μM). Mitochondrial stress was assessed 12 hours after drug treatment. For the mitochondrial stress test, 97.2 mL of Seahorse basal medium (Seahorse Bioscience, Billerica, MA, UAS) was heated to 37°C in a water bath and 1 mL of a stock solution was added. The stock solution was prepared by mixing the following solutions: 1 mL of 200 mM L-glutamine (Sigma), 1 mL of 100 mM pyruvate (Sigma), and 800 μL of 2.5 M glucose, adjusted to pH 7.4 with 1 M NaOH. Before testing, remove the probe card, add 1 mL of XF Carlibrant solution (Seahorse Bioscience) to the probe card, and place it in a 37°C constant temperature incubator without CO2 for overnight incubation. After drug treatment, remove the cells and wash the cells twice with seahorse basal medium preheated at 37°C to ensure that 500 μL of seahorse basal medium remains in each well, and place the cells in a 37°C constant temperature incubator without CO2 for 1 hour. During this period, remove the probe card from the incubator, add oligomycin, FCCP and Rotenone / antimycinA to the probe card, and then place the probe card in the Seahorse XFe analyzer for calibration. All drugs use 10 μM. The experiment is repeated 3-5 times.

[0186] The experimental results are shown in Table 2.

[0187] Table 2

[0188] Table 2 lists the effects of the drugs on basal cellular respiration, with the control group (DMSO-treated) set as 1. Statistically significant differences are indicated with an asterisk. The results show that terazosin and T1-T9 all have the activity to enhance mitochondrial respiration. T10 and T11 do not have mitochondrial activation activity. These experimental results are consistent with those in Figure 1. Both indicate that the newly synthesized isoquinoline derivatives (T1-T9) have the activity to activate Pgk1 and increase mitochondrial respiration.

[0189] Example 32: Determining whether a compound exerts its effect through Pgk1

[0190] Experimental conditions:

[0191] To further confirm the mitochondrial activation activity of the newly synthesized isoquinoline derivatives by targeting Pgk1, a cell line stably knocking down Pgk1 in SK-N-SH cells was constructed. The target sequence for human Pgk1 is GAGCTAAAGTTGCAGACAA. A BamHI restriction site was added to the 5' end of the knockdown sequence, an XbaI restriction site was added to the 3' end, and a loop was inserted in the middle. The resulting top strand was 5'-gatccGAGCTAAAGTTGCAGACAATTCAAGAGATTGTCTGCAACTTTAGCTCt-3', and the bottom strand was 5'-ctagaGAGCTAAAGTTGCAGACAATCTCTTGAATTGTCTGCAACTTTAGCTCg-3'. Both DNA oligos were phosphorylated at the 5' end and inserted into the plenti-u6 vector. The resulting Lenti-shPgk1 vector was transfected into HEK293T cells with pCAG-dR8.9 and pCMV-VSV-G at a molar ratio of 1:1:1, and the supernatant was collected after 72 hours. The resulting supernatant was subjected to ultracentrifugation to obtain a viral pellet, which was resuspended in 1 / 100 volume of PBS, allowed to stand for 24 hours, and then infected into polybrene-treated SH-SKN cells. After 48 hours of infection, the resulting virus-infected cells were inoculated into 96-well plates by limiting dilution to obtain a monoclonal cell line. The resulting monoclonal cell line was further expanded and cultured. Finally, western blot was used to further identify the knockdown efficiency of Pgk1. The results are shown in Figure 2, where A is an immunoblot experiment and B is a mitochondrial respiratory physiology experiment (seahorse assay). Figure 2 A shows that Pgk1 protein was significantly knocked down in the cell line stably expressing shPgk1. In the protein immunoblot results, GAPDH is a control for protein loading to ensure the same loading amount. Figure 2B compares the effects of T6 on mitochondrial respiration using this cell line. Seahorse's experimental conditions, dosing schedule, and other factors were identical to those in Example 31. The results showed that T6 (10 μM) activated both the basal and maximal respiration rates of normal cells (control); however, this activity was abolished in cells with shPgk1 knockdown. This suggests that T6's activation of mitochondrial metabolism is dependent on Pgk1. Similar results were observed with other compounds.

[0192] Example 33: Determination of the dose-effect relationship of the compound in in vitro biochemical experiments

[0193] Experimental conditions:

[0194] Human Pgk1 cDNA was cloned into the pEASYTM-E2 vector (TransGen Biotech) containing a His tag. Protein expression was induced in BL21 (DE3) chemically competent cells (TransGen Biotech) with 0.5 mM IPTG. After lysing the cells in extraction buffer (20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole, 5% glycerol, protease inhibitor cocktail, pH 7.9), the supernatant was purified using nickel beads (Adar Biotech). His-tagged Pgk1 was eluted using elution buffer (20 mM sodium phosphate, 500 mM NaCl, 80 mM imidazole, protease inhibitor cocktail, pH 7.9). Pgk1 activity can be measured by monitoring the rate of NADH production, as NADH should accumulate as the reaction proceeds. Purified Pgk1-His protein (2 μg / mL) was added to reaction buffer (20 mM Tris, 100 mM NaCl, 2 mM DTT, 0.1 mM MgSO₄, 10 mM NaHPO₄, pH 8.6) and substrates (1.6 mM GAP, 1 mM β-NAD, 1 mM ADP, 20 ng / μL GAPDH). Terazosin (positive control) and T6 were added to the reaction system at varying concentrations. After reacting at room temperature for 10 minutes, the reaction was performed using a microplate reader (Molecular Devices, SpectraMax i3x) at an absorbance wavelength of 339 nm, which allows for the detection of NADH levels.

[0195] The experimental results are shown in Figure 3, wherein A is the effect of terazosin on the enzyme activity of Pgk1 in cells, and B is the effect of compound T6 on the enzyme activity of Pgk1 in cells.

[0196] As shown in Figure 3A, as a positive control, the effects of terazosin 100μM, 20μM, 1μM, 0.1μM and 0.01μM on Pgk1 enzyme activity were detected. The results showed that high concentration (100μM) of terazosin inhibited Pgk1 enzyme activity, while low concentration (1μM) had the best effect in activating Pgk1. Figure 3B compared the effects of different concentrations of T6 on Pgk1 and found that various concentrations could not inhibit Pgk1. The optimal concentration for activating Pgk1 is 0.1μM. This result shows that T6 has no risk of inhibiting Pgk1. It may not have the effect of reducing brain tissue ATP in patients, and this effect reduces the risk of zosin drugs. T1-9 all had similar results.

[0197] Example 34: Determining the dose-effect relationship of a compound in cultured cells

[0198] Experimental conditions:

[0199] SK-N-SH cells were seeded into six empty plates at a density of 6 × 10 4 Cells were incubated under 5% O₂ for 12 hours before drug addition. Cells were harvested 48 hours later. Cellular ATP levels were measured using the Promega ATP assay kit. Experiments were repeated 5–7 times.

[0200] The experimental results are shown in Figure 4.

[0201] As shown in Figure 4, the positive controls, terazosin (TZ) and T6, were used at doses of 5, 10, and 50 μM. The optimal dose of TZ was 10 μM, and the optimal dose of T6 was 50 μM. However, both TZ and T6 at 50 μM showed a tendency to inhibit cell growth, suggesting that the biologically effective concentration of T6 should be closer to 10 μM.

[0202] Example 35: Determining the Effect of Compounds on Blood Glucose in Mice

[0203] Experimental conditions:

[0204] Wild-type mice (C57BL / 6) aged 3 to 4 months, housed under normal conditions, were intraperitoneally injected with various drugs at 0.1 mg / kg. A control group received saline. Following administration, the mice were fasted for 6 hours. Blood was collected from the tail for measurement of blood glucose levels. Nine mice were included in each experimental group.

[0205] The experimental results are shown in Figure 5.

[0206] As shown in Figure 5, the terazosin (TZ) group showed no hypoglycemic effect, while T1-9 had a significant hypoglycemic effect, indicating that this new class of isoquinoline derivatives has a higher activity in activating glucose metabolism.

[0207] Example 36: Determination of the hypoglycemic effect of the compound in diabetic model mice

[0208] Experimental conditions:

[0209] The gene-knockout db / db mouse is a classic model of hyperglycemia and obesity. Under normal feeding conditions, mice over 3 months old have developed hyperglycemia and obesity. After 6 hours of providing mice with only drinking water (fasting), blood was taken from the mouse's tail to measure blood sugar levels. Then different drugs were injected intraperitoneally, including normal saline (control), liraglutide (liraglutide) 0.6 mg / Kg, low concentration T6 (T6 low, 0.1 mg / Kg) and high concentration T6 (T6 high, 0.5 mg / Kg); after 4 hours, blood was taken from the mouse's tail to measure blood sugar levels. There were 6 mice in each experimental group. The blood sugar value (Vbefore ) and blood glucose level after administration (V after The effect of the drug on blood sugar (%) is calculated as follows:

[0210] (V before -V after )×100 / V before .

[0211] The experimental results are shown in Figure 6.

[0212] As shown in Figure 6, liraglutide, used as a positive control, demonstrated the best hypoglycemic efficacy. T6 also exhibited significant hypoglycemic efficacy at both low (0.1 mg / kg) and high (0.5 mg / kg) concentrations. This result further confirms the hypoglycemic efficacy of the novel isoquinoline derivatives when administered via intraperitoneal injection.

[0213] Intraperitoneal administration of novel isoquinoline derivatives has a hypoglycemic effect that lasts no longer than six hours. To confirm the long-term hypoglycemic effect of T6, we added T6 to the drinking water of mice for one week. Figure 7 shows the long-term effects of drinking water administration on mouse blood glucose.

[0214] Example 37: Determination of the long-term hypoglycemic and anti-obesity effects of the compound in diabetic mice

[0215] Experimental conditions:

[0216] Normally housed knockout db / db mice were treated with metformin (350 mg / kg), terazosin (0.3 mg / kg), or T6 (1.5 mg / kg) in their drinking water. After 7 days of treatment, changes in glycated hemoglobin and insulin levels in the mice's blood, as well as changes in body weight, were measured.

[0217] The experimental results are shown in FIG7 , wherein A is the glycated hemoglobin content, B is the insulin content, and C is the weight change of the mice.

[0218] As shown in Figure 7, compared to the control group, only the T6-treated group showed a decrease in glycated hemoglobin (HbA1c) levels. Insulin levels remained unchanged in all treatment groups. The T6-treated group showed a significant decrease in body weight, with the effects on body weight different from those seen in the terazosin and metformin-treated groups. These results suggest that long-term oral administration of T6 is more effective than terazosin and metformin in lowering blood sugar and also reduces body weight.

[0219] Example 38: Determining the Effects of Compounds on Dopamine Neurons in an MPTP-Induced Parkinson's Disease Mouse Model

[0220] Experimental conditions:

[0221] Three-month-old male mice were taken and raised normally. The acute MPTP model was established (see Sonsalla PK, Heikkila RE. The influence of dose and dosing interval on MPTP-induced dopaminergic neurotoxicity in mice. Eur J Pharmacol 1986; 129(3): 339-45.). Four doses were administered intraperitoneally. Each dose was 20 mg / kg body weight, with a 2-hour interval between each dose. MPTP was dissolved in saline, and the control group received an equal dose of saline. T2 was dissolved in DMSO and diluted with saline to the desired concentration. Therefore, equal doses of DMSO and saline were administered during the treatment. On the day of MPTP model establishment, mice were treated with T2 (treatment group) or DMSO (control group), and each group was subsequently treated with the drug daily. Mouse behavior was assessed using a rotarod (E103, UGO BASILE). First, at a fixed speed of 15 revolutions per minute (15rpm), mice were pre-trained for two consecutive days until they could stay on the rod for more than 60 seconds. On the 7th day after MPTP injection, mice were tested on a rotating rod in an accelerated mode (2-45rpm). The time mice continued on the rotating rod was recorded. This behavior was monitored by a video camera. After the experiment, mice were killed, striatum brain tissue was taken, and immunoblotting (Western Blot) experiment was performed. The protein specifically expressed in dopamine neurons is tyrosine hydroxylase (TH). Immunoblotting experiment was carried out with TH antibody to determine whether dopamine neurons were damaged. Terazosin (TZ, 0.1mg / Kg) was set as a positive control, and T2 tried three dosages: 0.1mg / Kg, 0.3mg / Kg, and 1mg / Kg.

[0222] The experimental results are shown in Figure 8, where A shows the effect of each drug on the motor ability of MPTP-induced Parkinson's disease model mice, B shows the effect of terazosin on the tyrosine hydroxylase content in MPTP-induced Parkinson's disease model mice, and C shows the effect of 0.1 mg / Kg dose of compound T2 on the tyrosine hydroxylase content in MPTP-induced Parkinson's disease model mice.

[0223] As shown in Figure 8 A, terazosin (TZ) treatment can improve the motor ability of mice under MPTP injury state. All three doses of T2 have a protective effect. Figure 8 B compares the tyrosine hydroxylase protein content of the control group, MPTP-treated group and terazosin-treated group. Striatal protein of 4 mice was randomly taken from each group. An immunoblotting experiment was performed. The statistical results are on the right. The results show that terazosin has the effect of protecting dopamine neurons. This group of experiments is a positive control. Figure 8 C is dopamine neurons, which is similar to the method of Figure 8 B. The results show that T2 has the effect of protecting dopamine neurons.

[0224] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An isoquinoline derivative, characterized in that: The general structural formula of the isoquinoline derivatives is as follows: In the above formula, ring A represents an optionally substituted five-membered oxygen-containing heterocyclic ring; X is O or C, Y is O or C, and X and Y contain at least one O; R 1 represents a hydrogen atom or a methyl group; R 2 represents a hydrogen atom or a methyl group; R 3 is a five-membered cycloalkyl group or a five-membered heterocyclic group containing one heteroatom, wherein the heteroatom is selected from an oxygen atom and a nitrogen atom; The structure of the isoquinoline derivatives is as follows:

2. A pharmaceutically acceptable salt of the isoquinoline derivative according to claim 1, comprising hydrochloride, sulfate, hydrobromide, nitrate, trifluoroacetate, acetate, phosphate, benzenesulfonate, p-toluenesulfonate, methanesulfonate, oxalate, tartrate, fumarate, maleate, succinate, malate, benzoic acid, citrate or lactate.

3. An isoquinoline derivative preparation, characterized in that: The active ingredient is the isoquinoline derivatives according to claim 1 and / or the pharmaceutically acceptable salts of the isoquinoline derivatives according to claim 2. The preparations are various types of pharmaceutical preparations prepared by combining the above-mentioned isoquinoline derivatives or the pharmaceutically acceptable salts of the above-mentioned isoquinoline derivatives with pharmaceutically acceptable excipients, such as water, alcohol, vegetable oil, starch, microcrystalline cellulose, lactose, gelatin, magnesium stearate, sodium alginate, maltodextrin, talc, cross-linked polyvinylpyrrolidone, gum and vaseline.

4. A phosphoglycerol kinase activator, characterized in that The activator is: the isoquinoline derivative according to claim 1, the pharmaceutically acceptable salt of the isoquinoline derivative according to claim 2, or the isoquinoline derivative preparation according to claim 3.

5. A drug for treating or preventing neurodegenerative diseases, characterized in that: The drug is: the isoquinoline derivative according to claim 1, the pharmaceutically acceptable salt of the isoquinoline derivative according to claim 2, or the isoquinoline derivative preparation according to claim 3.

6. A drug for treating or preventing metabolic diseases, characterized in that: The drug is: the isoquinoline derivative according to claim 1, the pharmaceutically acceptable salt of the isoquinoline derivative according to claim 2, or the isoquinoline derivative preparation according to claim 3.

7. A method for preparing the isoquinoline derivatives according to claim 1, characterized in that: The following synthetic routes or their combinations are included: Synthesis route A: Add malonic acid and piperidine to a solution of compound I to obtain compound II after reaction; compound II reacts under a catalyst and H2 atmosphere to obtain compound III; compound III first reacts with oxalyl chloride to generate acyl chloride, and then obtains compound IV under the catalysis of aluminum chloride; compound IV reacts with isoamyl nitrite under acidic conditions to obtain compound V; Compound V reacts with phosphorus oxychloride to obtain compound VI; compound VI reacts with an amino protecting agent in N-methylpyrrolidone to obtain compound VII; compound VII, compound XI, dioxane, Pd2(dba)3, RuPhos and cesium carbonate react together to obtain compound VIII; compound VIII removes the Boc protecting group to obtain compound IX; compound IX reacts with R 3 -COOH reaction to prepare compound X; compound X reacts with trifluoroacetic acid to remove amino protection to prepare the isoquinoline derivative compound T; the reaction route is as follows: Synthesis route B: Compound XI reacts with R in a solvent 3 -COOH, O-(7-nitrobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine are reacted to obtain compound XII; compound XII is deprotected by removing the Boc protecting group to obtain compound XIII; compound XIII is reacted with compound VII prepared by reference to synthesis route A, dioxane, Pd2(dba)3, RuPhos and cesium carbonate to obtain compound X; compound X is reacted with trifluoroacetic acid to remove the amino protection to obtain the isoquinoline derivative compound T; the reaction route is as follows: Synthetic route C: Compound VI prepared by referring to synthetic route A is reacted with ammonia water to obtain compound XIV; compound XIV is reacted with an amino protecting agent to obtain compound VII; compound VII is used to obtain the isoquinoline derivative compound T by referring to the method of synthetic route A; the reaction route is as follows: The R of the compounds involved in the synthetic routes A, B and C 4 The group is an amino protecting group.

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