阅读说明：本技术 一种1,2,4-噁二唑-吡啶类化合物及其在治疗阿尔茨海默症中的应用 (1,2, 4-oxadiazole-pyridine compound and application thereof in treating Alzheimer's disease ) 是由 李增 刘彤彤 杜吉宇 邢思琪 陈世明 于 2021-07-19 设计创作，主要内容包括：本发明属于药物化合物制药应用技术领域,具体涉及一种1,2,4-噁二唑-吡啶类化合物及其在治疗阿尔茨海默症中的应用。该1,2,4-噁二唑-吡啶类化合物结构式如通式A所示：其中R为正丙胺、正丁胺、异丁胺、环丙胺、环戊胺、苯胺、3-甲氧基苯胺、4-氟苯胺、3-氟苯胺、4-氯苯胺、3-氯苯胺、4-溴苯胺、3,4-二氟苯胺、4-三氟甲基苯胺、邻甲基苯胺、间甲基苯胺、对甲基苯胺、苄胺、4-氟苄胺、4-三氟甲基苄胺、3-氟苄胺中的任意一种。该化合物具备抑制丁酰胆碱酯酶活性和神经保护作用,表明该类化合物有潜力可以发展成为治疗阿尔兹海默症的药物。(The invention belongs to the technical field of pharmaceutical application of pharmaceutical compounds, and particularly relates to a 1,2, 4-oxadiazole-pyridine compound and application thereof in treating Alzheimer's disease. The structural formula of the 1,2, 4-oxadiazole-pyridine compound is shown as a general formula A: wherein R is n-propylamine, n-butylamine, isobutylamine, cyclopropylamine, cyclopentylamine, aniline, 3-methoxyaniline, 4-fluoroaniline, 3-fluoroaniline, 4-chloroaniline, 3-Any one of chloroaniline, 4-bromoaniline, 3, 4-difluoroaniline, 4-trifluoromethylaniline, o-toluidine, m-toluidine, p-toluidine, benzylamine, 4-fluorobenzylamine, 4-trifluoromethylbenzylamine, and 3-fluorobenzylamine. The compound has the effects of inhibiting butyrylcholine esterase activity and protecting nerves, and shows that the compound has potential to be developed into a medicine for treating Alzheimer's disease.)

1. A1, 2, 4-oxadiazole-pyridine compound is characterized in that the structural formula is shown as a formula (A):

wherein R is any one of n-propylamine, n-butylamine, isobutylamine, cyclopropylamine, cyclopentylamine, aniline, 3-methoxyaniline, 4-fluoroaniline, 3-fluoroaniline, 4-chloroaniline, 3-chloroaniline, 4-bromoaniline, 3, 4-difluoroaniline, 4-trifluoromethylaniline, o-methylaniline, m-methylaniline, p-methylaniline, benzylamine, 4-fluorobenzylamine, 4-trifluoromethylbenzylamine and 3-fluorobenzylamine.

2. Use of a 1,2, 4-oxadiazole-pyridine compound according to claim 1 in a medicament for inhibiting butyrylcholinesterase activity.

3. Use of the 1,2, 4-oxadiazole-pyridine compound of claim 1 for the preparation of a neuroprotective drug.

4. Use of a 1,2, 4-oxadiazole-pyridine compound according to claim 1 for the preparation of a medicament for the treatment of alzheimer's disease.

5. The use of claim 4, wherein the medicament is any one of an injection, a tablet, a pill, a capsule, a suspension or an emulsion.

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a 1,2, 4-oxadiazole-pyridine compound and application thereof in preparation of a neuroprotective drug and a drug for treating Alzheimer's disease.

Background

Alzheimer's disease is one of the most common neurodegenerative diseases and is clinically characterized by the appearance of cognitive dysfunction, especially memory loss, in patients. In recent years, the prevalence has increased year by year with the aging of the global population. By 2050, AD is expected to reach 1.3 billion worldwide. Drugs currently approved by the FDA for the treatment of AD often provide temporary or incomplete relief of symptoms, with severe side effects. Therefore, there is an urgent need to develop more effective therapeutic drugs for alzheimer's disease.

The pathogenesis of AD is extremely complex, and to date there is a definite cause. Studies that have been performed to date have shown that the main features of AD are neuronal loss and reduced neurotransmitter levels, amyloid peptide (a β) deposition and neurofibrillary tangles (NFT). Wherein the decrease in neurotransmitter levels is primarily manifested by a massive loss of acetylcholine (ACh) in the brain. ACh is centrally located and studies have shown that a decrease in cognitive function in AD patients correlates with a decrease in ACh levels. AD can therefore be treated by inhibiting a decrease in ACh levels. ACh is mainly hydrolyzed by acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), which are mainly expressed by neurons, but BuChE activity is associated with neuritic plaques, neurofibrillary tangles and glial cells. In the brains of patients in the late phase of AD, AChE levels are significantly reduced and BuChE activity is increased, thus exacerbating A β toxicity. In addition, BuChE can replace AChE to maintain loss of AChE in AChE knockout mouse models. Classical AChE inhibitors and non-selective cholinesterase inhibitors have been reported to have peripheral cholinergic side effects, but not in selective BuChE inhibitors. This suggests that the development of selective BuChE inhibitors is an important approach to the treatment of alzheimer's disease in AD.

In addition, Alzheimer's disease, a neurological disease, H₂O₂The development of this disease is greatly accelerated by neurotoxicity caused by induced oxidative damage, and the development of compounds with neuroprotective effects may have therapeutic effects on alzheimer's disease. Because the pathogenic mechanism of AD is complex and the factors are related, the traditional single-molecule targeted therapy strategy has little beneficial effect on AD and has higher clinical failure rate. Therefore, multi-target ligand (MTDLs) therapeutic strategies have many advantages for AD, a complex disease.

Disclosure of Invention

In order to solve the above-mentioned problems, an object of the present invention is to provide a 1,2, 4-oxadiazole-pyridine compound.

The technical scheme adopted by the invention is as follows: a1, 2, 4-oxadiazole-pyridine compound has a structural formula shown as a formula (A):

wherein R is any one of n-propylamine, n-butylamine, isobutylamine, cyclopropylamine, cyclopentylamine, aniline, 3-methoxyaniline, 4-fluoroaniline, 3-fluoroaniline, 4-chloroaniline, 3-chloroaniline, 4-bromoaniline, 3, 4-difluoroaniline, 4-trifluoromethylaniline, o-methylaniline, m-methylaniline, p-methylaniline, benzylamine, 4-fluorobenzylamine, 4-trifluoromethylbenzylamine and 3-fluorobenzylamine.

The preparation route of the 1,2, 4-oxadiazole-pyridine compound is as follows:

the preparation routes are mainly intended to illustrate the invention without imposing any limitation thereon.

i. According to the method reported by the literature, 2-chloro-4-cyanopyridine and 4-trifluoromethoxy aniline are subjected to nucleophilic substitution reaction, water is added for precipitation, and then petroleum ether is used for washing to obtain a compound a;

reaction of compound a with hydroxylamine hydrochloride in K₂CO₃Reacting under the action of the catalyst to generate a compound b;

iii, reacting the compound b with 4-nitrobenzoyl chloride to obtain a compound c;

the compound c is subjected to reduction reaction to obtain a compound d;

v. the compound d is purified by column chromatography and then reacts with chloroacetyl chloride to obtain an intermediate e.

And vi, dissolving the intermediate e in absolute ethyl alcohol, reacting with amines with various substituents (75 ℃), and purifying by column chromatography to obtain the compounds f1-f 21. Wherein R is any one of n-propylamine, n-butylamine, isobutylamine, cyclopropylamine, cyclopentylamine, aniline, 3-methoxyaniline, 4-fluoroaniline, 3-fluoroaniline, 4-chloroaniline, 3-chloroaniline, 4-bromoaniline, 3, 4-difluoroaniline, 4-trifluoromethylaniline, o-methylaniline, m-methylaniline, p-methylaniline, benzylamine, 4-fluorobenzylamine, 4-trifluoromethylbenzylamine and 3-fluorobenzylamine.

The invention also aims to provide the application of the 1,2, 4-oxadiazole-pyridine compound in inhibiting butyrylcholinesterase (BuChE) activity.

The invention also aims to provide the application of the 1,2, 4-oxadiazole-pyridine compound in preparing the anti-neuritis medicine.

Based on the application of the 1,2, 4-oxadiazole-pyridine compound in inhibiting butyrylcholinesterase (BuChE) activity and preparing anti-neuritis drugs, the invention also provides the application of the 1,2, 4-oxadiazole-pyridine compound in preparing drugs for treating Alzheimer's disease.

Further, the medicine is any one of injection, tablet, pill, capsule, suspending agent or emulsion.

The invention has the advantages that:

1. the 1,2, 4-oxadiazole-pyridine compounds have an inhibiting effect on the activity of BuChE, and show that the compounds have potential to be developed into effective BuChE inhibitors.

2. The 1,2, 4-oxadiazole-pyridine compound can well improve H₂O₂The survival rate of the induced PC12 cells shows that the compound can be applied as a neuroprotective medicament.

3. The compounds of the present invention have the potential to be developed into drugs for treating alzheimer's disease by inhibiting BuChE activity and/or by neuroprotective pathways.

Drawings

FIGS. 1-5 are graphs showing the results of various data in the Morris water maze test for mice in example 24. Wherein the content of the first and second substances,

FIG. 1 is a graph of the effect of varying concentrations of compound f9 on mouse body weight over a 7 day dosing period;

FIG. 2 is a result of difference in swimming speed between the drug group and the normal group;

FIG. 3 shows the time at which the drug group and normal group mice found the platform;

FIG. 4 is the results of residence time in the target quadrant for the drug and normal group mice after the platform was removed;

fig. 5 shows the results of the number of crossings of the drug group and normal group mice on the virtual platform after the platform was removed.

FIG. 6 is a representative trace plot of the Morris water maze test for mice from example 24. As can be seen from the figure, the time taken for mice treated with a dose of 30mg/kg to enter the quadrant of the platform and the number of times to enter the zone after removing the platform was significantly better than the model group, except for the normal group.

Detailed Description

Unless otherwise indicated, the terms used herein have the meanings that are conventionally understood by those skilled in the art.

The technical scheme of the invention is more specifically explained by combining the following embodiments:

example 1

Synthesis of compound f 1:

compound e (294mg, 0.6mM) was dissolved in 80mL of absolute ethanol, followed by addition of n-propylamine (457mg, 4.8mM) and triethylamine (101mg, 1.2mM) and refluxing at 75 ℃ for 8-12 h. The progress of the reaction was checked by TLC. After the reaction, the reaction solvent was removed. The compound f1 is obtained after purification by column chromatography with ethyl acetate and petroleum ether (1: 4). Compound f1 was a white solid in 72% yield.

¹H NMR(500MHz,DMSO-d₆)δ9.66(s,1H),8.36(d,J＝5.3Hz,1H),8.13(d,J＝8.8Hz,2H),7.93(d,J＝8.8Hz,2H),7.85(d,J＝9.1Hz,2H),7.56(s,1H),7.38–7.26(m,3H),3.34(s，2H)，2.53(d，J＝3.3Hz,2H),1.47(h,J＝7.3Hz,2H)，0.90(t，J＝7.4Hz,3H)；¹³C NMR(126MHz,DMSO)δ12.18,23.10,51.54,53.41,109.27,111.84,117.92,119.55,119.71,119.75,121.73,122.07,129.52,135.11,141.02,142.22，143.77,149.02，156.68，167.48,171.83,176.06。

HRMS(ESI)m/z[M+H]⁺:513.18137calcd for C₂₅H₂₃F₃N₆O₃:513.18518。

Example 2

Synthesis of compound f 2:

the procedure is as in example 1, except that the R group is replaced by n-butylamine, giving compound f2 as a white solid in 68% yield.

¹H NMR(500MHz,DMSO-d₆)δ9.66(s，1H)，8.36(d，J＝5.3Hz，1H)，8.12(d，J＝8.8Hz，2H)，7.93(d，J＝8.7Hz，2H)，7.85(d，J＝9.1Hz，2H)，7.56(s，1H)，7.41–7.25(m，3H)，3.34(s，2H)，2.55(t，J＝7.0Hz，2H)，1.43(td，J＝13.7，12.6，6.1Hz，2H),1.34(h，J＝7.3Hz,2H),0.89(t，J＝7.3Hz，3H)；¹³C NMR(126MHz，DMSO)δ14.36,20.34,32.12,49.27,53.47,109.27,111.84,117.91,119.55，119.74,121.73,122.06,123.99，129.51,135.11,141.02,142.22,143.77，149.01,156.68,167.48,171.82,176.05。

HRMS(ESI)m/z[M+H]⁺:527.19738calcd for C₂₆H₂₅F₃N₆O₃:527.20117。

Example 3

Synthesis of compound f 3:

the procedure is as in example 1, except that the R group is replaced by isobutylamine, giving compound f3 as a white solid in 67% yield.

¹H NMR(500MHz,DMSO-d₆)δ9.67(s,1H),8.37(d,J＝5.2Hz,1H),8.14(d,J＝8.9Hz,2H),7.93(d,J＝8.8Hz,2H),7.85(d,J＝9.2Hz,2H),7.57(s,1H),7.36(dd,J＝5.2,1.4Hz,1H),7.30(d,J＝8.0Hz,2H),3.34(s,4H),2.36(d,J＝6.7Hz,2H),1.70(dp,J＝13.3,6.7Hz,1H),0.91(d,J＝6.6Hz,6H)；¹³C NMR(126MHz,DMSO)δ21.07,28.47,53.61,57.74,109.28,111.88,117.94,119.57,119.71,119.73,121.74,122.11,129.59,135.14,141.02,142.23,142.25,143.76,149.08,156.70,167.51,171.84,176.09,179.25。

HRMS(ESI)m/z[M+H]⁺:527.19738calcd for C₂₆H₂₅F₃N₆O₃:527.20062。

Example 4

Synthesis of compound f 4:

the procedure is as in example 1, except that the R group is replaced by cyclopropylamine, giving compound f4 as a white solid in 64% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.25(s,1H),9.66(s，1H),8.37(s,1H),8.13(d,J＝8.2Hz,2H),7.89(dd,J＝43.7,8.5Hz,4H),7.56(s,1H),7.43–7.23(m,3H),2.20(s,1H),0.36(d,J＝36.4Hz,4H)；¹³C NMR(126MHz,DMSO)δ6.55,30.51,53.25,109.27,111.86,117.89,119.57,119.71,119.78,121.74,122.84,129.53,135.13,141.02,142.23,143.89,149.04,156.69,167.50,171.86,176.09。

HRMS(ESI)m/z[M+H]⁺:511.16608calcd for C₂₅H₂₁F₃N₆O₃:511.16913。

Example 5

Synthesis of compound f 5:

the procedure is as in example 1, except that the R group is replaced by cyclopentylamine, giving compound f5 as a white solid in 65% yield.

¹H NMR(500MHz,DMSO-d6)δ9.61(s,1H),8.30(d,J＝5.3Hz,1H),8.08(d,J＝8.9Hz,2H),7.86(d,J＝8.8Hz,2H),7.78(d,J＝9.2Hz,2H),7.50(s,1H),7.29(dd,J＝5.3,1.4Hz,1H)，7.23(d,J＝8.6Hz,2H),3.34(s,3H),3.02(p,J＝6.2Hz,1H),1.74-1.53(m,4H),1.47-1.28(m,4H)；¹³C NMR(126MHz,DMSO)δ23.90,32.55,51.84,59.55,109.27,111.88,118.02,119.58,119.71,119.78,121.74,122.09,129.59,135.13,141.02,142.25,143.69,149.07,156.71,167.52,171.37,176.08。

HRMS(ESI)m/z[M+H]⁺:539.19738calcd for C₂₇H₂₅F₃N₆O₃:539.20026。

Example 6

Synthesis of compound f 6:

the procedure is as in example 1, except that the R group is replaced by aniline, giving compound f6 as a white solid in 62% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.48(s,1H),9.67(s,1H),8.37(d,J＝5.2Hz,1H),8.15(d,J＝8.8Hz,2H),7.93(d,J＝8.9Hz,2H),7.85(d,J＝9.1Hz,2H),7.57(t,J＝1.1Hz,1H),7.36(dd,J＝5.3,1.4Hz,1H),7.30(d,J＝8.1Hz,2H),7.13–7.09(m,2H),6.64–6.57(m,3H),3.95(s,2H)；¹³C NMR(126MHz,DMSO)δ47.88,109.28,111.90,112.81,116.98,118.00,119.60,119.71,119.91,121.74,122.12,129.39,129.59,135.16,141.01,142.27,143.96,148.70,149.09,156.71,167.53,170.75,176.11。

HRMS(ESI)m/z[M+H]⁺:547.16608calcd for C₂₈H₂₁F₃N₆O₃:547.16919。

Example 7

Synthesis of compound f 7:

the procedure is as in example 1, except that the R group is replaced by 3-methoxyaniline, giving compound f7 as a white solid in 52% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.48(s,1H),9.67(s,1H),8.37(d,J＝5.3Hz,1H),8.18–8.13(m,2H),7.95–7.90(m,2H),7.87–7.82(m,2H),7.57(s,1H),7.36(dd,J＝5.2,1.4Hz,1H),7.30(d,J＝8.6Hz,2H),7.00(t,J＝8.4Hz,1H),6.23(dd，J＝7.5,2.0Hz,1H),6.20–6.17(m,2H),6.07(t,J＝6.2Hz,1H),3.93(d,J＝6.0Hz,2H),3.67(s,3H)；¹³C NMR(126MHz,DMSO)δ47.88,55.13,98.72,102.42,105.82,109.28,111.89,118.00,119.58,119.71,119.89,121.74,122.11,129.60,130.14,135.15,141.02,142.25,143.95,149.10,150.08,156.71,160.77,167.53,170.69,176.10。

HRMS(ESI)m/z[M+H]⁺:577.17664 calcd for C₂₉H₂₃F₃N₆O₄:577.17950。

Example 8

Synthesis of compound f 8:

the procedure is as in example 1, except that the R group is replaced by 3-fluoroaniline, giving compound f8 as a white solid in 47% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.53(s,1H),9.67(s,1H),8.38(d,J＝5.3Hz,1H),8.15(d,J＝8.4Hz,2H),7.92(d,J＝8.2Hz,2H),7.85(d,J＝8.6Hz,2H),7.57(s,1H),7.36(d,J＝5.2Hz,1H),7.30(d,J＝8.5Hz,2H),7.11(q,J＝7.9Hz,1H),6.40(ddt,J＝24.0,16.1,7.9Hz,4H),3.98(s,2H)；¹³C NMR(126MHz,DMSO)δ47.49,102.81,102.98,108.96,109.26,111.90,118.03,119.59,119.89,122.12,129.62,130.74,130.82,135.14,141.01,142.25,143.92,149.10,150.86,150.95,156.70,162.91,164.89,167.52,170.25,176.09。

HRMS(ESI)m/z[M+H]⁺:565.15666 calcd for C₂₈H₂₀F₄N₆O₃:565.15961。

Example 9

Synthesis of compound f 9:

the procedure is as in example 1, except that the R group is replaced by 4-fluoroaniline, giving compound f9 as a white solid in 56% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.48(s,1H),9.67(s,1H),8.37(d,J＝5.3Hz,1H),8.14(d,J＝8.8Hz,2H),7.93(d,J＝8.8Hz,2H),7.86(d,J＝9.2Hz,2H),7.57(s,1H),7.35(dd，J＝5.2,1.4Hz,1H),7.30(d,J＝8.6Hz,2H),6.97(t，J＝8.9Hz,2H),6.63(dd，J＝9.0，4.5Hz,2H),6.03(t,J＝6.1Hz,1H),3.94(d,J＝5.8Hz,2H)；¹³C NMR(126MHz,DMSO)δ48.28,109.26,113.53,113.59,115.67,115.85,118.00,119.56,119.71,119.87,121.73,122.07,129.54,135.11,141.02,142.23,143.91,145.42,149.03,154.24,156.08,156.68,167.50,170.62,176.05。

HRMS(ESI)m/z[M+H]⁺:565.15666calcd for C₂₈H₂₀F₄N₆O₃:565.15985。

Example 10

Synthesis of compound f 10:

the procedure is as in example 1, except that the R group is replaced by 4-chloroaniline to give compound f10 as a white solid in 54% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.50(s,1H),9.66(s,1H),8.36(d,J＝5.2Hz,1H),8.13(d,J＝8.5Hz,2H),7.93(d,J＝8.7Hz,2H),7.89–7.83(m,2H),7.56(s,1H),7.36–7.26(m,3H),7.17–7.12(m,2H),6.70–6.63(m,2H),6.28(t,J＝6.1Hz,1H),3.97(d,J＝6.1Hz,2H)；¹³C NMR(126MHz,DMSO)δ47.69,109.26,111.82,114.16,117.68,118.01,119.54,119.71,119.84,120.24,121.74,122.01,123.77,129.05,129.50,135.09,141.02,142.23,143.88,147.71,148.96,156.66,167.48,170.28,176.01。

HRMS(ESI)m/z[M+H]⁺:581.12711 calcd for C₂₈H₂₀ClF₃N₆O₃:581.13049。

Example 11

Synthesis of compound f 11:

the procedure is as in example 1, except that the R group is replaced by 3-chloroaniline, giving compound f11 as a white solid in 45% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.53(s,1H),9.67(s,1H),8.38(d,J＝5.2Hz,1H),8.16(d,J＝8.8Hz,2H),7.92(d,J＝8.8Hz,2H),7.85(d,J＝9.1Hz,2H),7.57(s,1H),7.36(dd,J＝5.3,1.4Hz,1H),7.30(d,J＝8.6Hz,2H),7.11(t,J＝8.0Hz,1H),6.65(t,J＝2.1Hz,1H),6.58(ddd,J＝11.1,7.7,2.1Hz,2H),6.41(t,J＝6.2Hz,1H),3.98(d,J＝6.2Hz,2H)；¹³C NMR(126MHz,DMSO)δ21.07,28.47,53.61,57.74,109.28,111.88,117.94,119.57,119.71,119.73,121.74,122.11,129.59,135.14,141.02,142.25,143.76,149.08,156.70,167.51,171.84,176.09。

HRMS(ESI)m/z[M+H]⁺:581.12711 calcd for C₂₈H₂₀ClF₃N₆O₃:581.13025。

Example 12

Synthesis of compound f 12:

the procedure is as in example one, except that the R group is replaced with 4-bromoaniline, giving compound f12 as a white solid in 48% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.51(s,1H),9.67(s,1H),8.37(d,J＝5.3Hz,1H),8.14(d,J＝8.6Hz,2H),7.92(d,J＝8.7Hz,2H),7.88–7.81(m,2H),7.57(s,1H),7.35(dd,J＝5.2,1.4Hz,1H),7.30(d,J＝8.6Hz,2H),7.27–7.23(m,2H),6.63–6.57(m,2H),6.31(s,1H),3.96(d,J＝3.3Hz,2H)；¹³C NMR(126MHz,DMSO)δ47.58,107.58,109.27,111.86,114.73,118.01,119.57,119.71,119.87,121.74,122.08,123.76,129.56,131.88,135.12,141.02,142.24,143.91,148.10,149.04,156.69,167.50,170.28,176.06。

HRMS(ESI)m/z[M+H]⁺:625.07659 calcd for C₂₈H₂₀BrF₃N₆O₃:625.08014。

Example 13

Synthesis of compound f 13:

the procedure is as in example 1 except that the radical R is 3, 4-difluoroaniline, and a white solid, compound f13, is obtained in 43% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.52(s,1H),9.66(s,1H),8.37(d,J＝5.2Hz,1H),8.18–8.10(m,2H),7.95–7.89(m,2H),7.89–7.82(m,2H),7.57(t,J＝1.1Hz,1H),7.35(dd,J＝5.3,1.4Hz,1H),7.30(d,J＝8.6Hz,2H),7.21–7.12(m,1H),6.63(ddd,J＝13.6,6.8,2.8Hz,1H),6.42(d,J＝8.9Hz,1H),6.30(s,1H),4.07–3.92(m,2H)；¹³C NMR(126MHz,DMSO)δ47.78,100.91,101.07,108.20,109.26,111.84,117.68,117.79,117.92,118.03,119.56,119.71,119.84,121.73,122.04,123.76,129.54,135.10,141.01,142.23,142.78,142.88,143.88,146.42,146.49,149.00,149.38,149.49,151.30,151.41,156.67,167.49,170.12,176.03。

HRMS(ESI)m/z[M+H]⁺:583.14723 calcd for C₂₈H₁₉F₅N₆O₃:583.15039。

Example 14

Synthesis of compound f 14:

the procedure is as in example 1, except that the radical R is 4-trifluoromethylaniline, and a white solid, compound f14, is obtained in 48% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.57(s,1H),9.67(s,1H),8.38(d,J＝5.3Hz,1H),8.16(d,J＝8.5Hz,2H),7.92(d,J＝8.7Hz,2H),7.88–7.81(m,2H),7.57(s,1H),7.42(d,J＝8.5Hz,2H),7.36(d,J＝5.2Hz,1H),7.30(d,J＝8.6Hz,2H),6.79(t,J＝6.2Hz,1H),6.73(d,J＝8.6Hz,2H),4.05(d,J＝6.2Hz,2H)；¹³C NMR(126MHz,DMSO)δ47.01,109.27,111.90,112.20,116.38,116.62,118.06,119.60,119.90,122.12,126.69,126.73,129.63,135.15,141.02,142.26,143.92,149.11,151.91,156.71,167.54,169.91,176.09。

HRMS(ESI)m/z[M+H]⁺:615.15346 calcd for C₂₈H₁₉F₅N₆O₃:615.15649。

Example 15

Synthesis of compound f 15:

the procedure is as in example 1, except that the R group is o-toluidine, giving compound f15 as a white solid in 43% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.47(s,1H),9.67(s,1H),8.37(d,J＝5.3Hz,1H),8.14(d,J＝8.4Hz,2H),7.93(d,J＝8.4Hz,2H),7.85(d,J＝8.6Hz,2H),7.57(s,1H),7.36(d,J＝5.4Hz,1H),7.30(d,J＝8.5Hz,2H),7.02(s,2H),6.57(t,J＝7.3Hz,1H),6.44(d,J＝8.2Hz,1H),5.37(t,J＝6.0Hz,1H),4.00(d,J＝5.9Hz,2H),2.18(s,3H)；¹³C NMR(126MHz,DMSO)δ17.99,48.04,109.29,109.72,111.87,117.01,118.01,119.57,119.71,119.92,121.73,122.10,122.52,127.28,129.57,130.32,135.12,141.02,142.24,143.90,146.35,149.07,156.70,167.51,170.76,176.07。

HRMS(ESI)m/z[M+H]⁺:561.18173 calcd for C₂₉H₂₃F₃N₆O₃:561.18457。

Example 16

Synthesis of compound f 16:

the procedure is as in example 1, except that the radical R is meta-methylaniline, giving compound f16 as a white solid in 58% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.47(s,1H),9.68(s,1H),8.37(s,1H),8.15(t,J＝8.3Hz,2H),7.94(t,J＝8.1Hz,2H),7.91–7.81(m,2H),7.57(s,1H),7.32(dt,J＝16.4,6.6Hz,3H),7.01(d,J＝7.5Hz,1H),6.61–6.35(m,3H),5.97(s,1H),3.94(d,J＝6.2Hz,2H),2.22(dd,J＝9.5,6.2Hz,3H)；¹³C NMR(126MHz,DMSO)δ21.81,47.94,109.28,110.01,112.27,113.58,117.97,119.57,119.71,119.89,121.65,121.73,122.09,129.27,129.55,135.12,138.36,141.02,142.25,143.95,148.69,149.05,156.70,167.51,170.79,176.07。

HRMS(ESI)m/z[M+H]⁺:561.18173 calcd for C₂₉H₂₃F₃N₆O₃:561.18488。

Example 17

Synthesis of compound f 17:

the procedure is as in example 1, except that the radical R is p-methylaniline, giving compound f17 as a white solid in 63% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.44(s,1H),9.66(s,1H),8.35(d,J＝5.3Hz,1H),8.12(d,J＝8.6Hz,2H),7.93(d,J＝8.5Hz,2H),7.90–7.83(m,2H),7.56(s,1H),7.33(dd,J＝5.2,1.4Hz,1H),7.29(d,J＝8.5Hz,2H),6.95(d,J＝8.0Hz,2H),6.58(d,J＝8.3Hz,2H),5.86(s,1H),3.93(s,2H)，2.17(s,3H)；¹³C NMR(126MHz,DMSO)δ20.48,48.29,109.27,111.80,112.98,117.69，117.97，119.53,119.72,119.82,121.74,121.98,123.77,125.51,129.44,129.82,135.08,141.02,142.22,143.88,146.38,148.91,156.65,167.46,170.82,175.99。

HRMS(ESI)m/z[M+H]⁺:561.18173 calcd for C₂₉H₂₃F₃N₆O₃:561.18488。

Example 18

Synthesis of compound f 18:

the procedure is as in example 1, except that the radical R is benzylamine, giving compound f18 as a white solid in 64% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.28(s,1H),9.66(s,1H),8.35(d,J＝5.3Hz,1H),8.10(d,J＝8.4Hz,2H),7.93(d,J＝8.4Hz,2H),7.90–7.83(m,2H),7.55(s,1H),7.41(d,J＝7.0Hz,2H),7.39–7.31(m,3H),7.27(dd，J＝18.1,8.0Hz,3H),3.80(s,2H),3.37(s,2H)；¹³C NMR(126MHz，DMSO)δ52.52,53.02,109.27，111.80,117.90,119.53,119.72,122.00,127.20，128.51,128.66,129.42,135.07,140.68,141.03,142.21,143.78,148.92,156.65,167.45,171.43,175.99。

HRMS(ESI)m/z[M+H]⁺:561.18173 calcd for C₂₉H₂₃F₃N₆O₃:561.18439。

Example 19

Synthesis of compound f 19:

the procedure is as in example 1, except that the radical R is 4-fluorobenzylamine, giving compound f19 as a white solid in 57% yield.

¹H NMR(500MHz,DMSO-d₆)δ10.28(s,1H),9.67(s,1H),8.37(d,J＝5.3Hz,1H),8.13(d,J＝8.9Hz,2H)，7.97–7.90(m，2H),7.89–7.84(m，2H)，7.56(s,1H),7.43(dd,J＝8.5,5.7Hz,2H),7.35(dd，J＝5.3,1.4Hz,1H),7.30(d,J＝8.6Hz,2H),7.16(t，J＝8.9Hz,2H),3.77(s,2H),3.35(s,2H)；¹³C NMR(126MHz,DMSO)δ52.16,52.44,109.27，111.84,115.25,115.42,117.91,119.55,119.71,119.79,121.74,122.07,123.77,129.48,130.35,130.41,135.11,136.85,136.87,141.03,142.22,143.82,149.01,156.68,160.69,162.62,167.48,171.45,176.06。

HRMS(ESI)m/z[M+H]⁺:579.17231 calcd for C₂₉H₂₂F₃N₆O₃:579.17523。

Example 20

Synthesis of compound f 20:

the procedure is as in example 1, except that the radical R is 4-trifluoromethylbenzylamine, giving compound f20 as a white solid in 49% yield.

¹H NMR(500MHz,DMSO-d6)δ10.29(s,1H),9.67(s,1H),8.37(d,J＝5.3Hz,1H),8.14(d,J＝8.6Hz,2H),7.92(d,J＝8.7Hz,2H),7.85(d,J＝9.1Hz,3H),7.70(d,J＝8.1Hz,2H),7.62(d,J＝7.9Hz,2H),7.57(s,1H),7.36(d,J＝5.3Hz,1H),7.30(d,J＝8.6Hz,2H),3.88(s,2H),3.37(s,2H)；13C NMR(126MHz,DMSO)δ52.36,52.50,109.27,111.88,117.93,119.57,119.71,119.83,121.74,122.11,125.49,125.52,127.76,128.01,129.15,129.53,135.14,141.02,142.24,143.85,145.82,149.08,156.70,167.51,171.40,176.10。

HRMS(ESI)m/z[M+H]+:629.16911 calcd for C30H22F6N6O3:629.17194。

Example 21

Synthesis of compound f 21:

the procedure is as in example 1, except that the radical R is 3-fluorobenzylamine, giving compound f21 as a white solid in 51% yield.

¹H NMR(500MHz,DMSO-d6)δ10.29(s,1H),9.67(s,1H),8.37(d，J＝5.3Hz,1H),8.14(d,J＝8.8Hz,2H),7.94(d,J＝8.9Hz,2H),7.85(d,J＝9.1Hz,2H),7.57(s,1H),7.41–7.33(m,2H),7.30(d,J＝8.6Hz,2H),7.23(dd,J＝15.6,8.5Hz,2H),7.07(td,J＝8.2,2.3Hz,1H),3.80(s,2H)；13C NMR(126MHz,DMSO)δ52.33,52.47,109.28,111.89,113.84,114.01,114.92,115.09,117.94,119.58,119.71,119.86,121.74，122.11，124.45，124.47，129.53，130.49，130.56，135.14，141.02，142.24，143.85,143.93,143.98,149.08,156.70,161.84，163.77,167.51,171.45,176.10。

HRMS(ESI)m/z[M+H]+:579.17231 calcd for C29H22F4N6O3:579.17487。

Example 22: inhibitory assay for BuChE Activity

1) Preparation of compound solution: using compounds f1-f21 as test compounds, the test compounds were prepared as solutions (solvent: DMSO) at a concentration of 100. mu.M, stored at low temperature of 20 ℃ and diluted to the desired concentration with the desired solvent or cell culture medium at the time of use.

2) Kinase inhibition experiments

Test compounds dissolved in DMSO were diluted to different concentrations with PB (phosphate buffered saline) solutions and then added to 48 well plates (100 μ L per well) before incubation with 100 μ L of AChE or BuChE at 37 ℃. After 20min, 100. mu.L of substrate (iodothioacetylcholine or S-butyrylthiocholine iodide) and 100. mu.L of PB solution were added to 48-well plates. Then 100. mu.L of a chromogenic substance (5, 5-dithiobis- (2-nitrobenzoic acid), DTNB, Ellman's reagent) was added and incubated for 5min, followed by absorbance detection at 419nm using a multifunctional microplate reader.

IC₅₀Defined as the concentration of compound that inhibited 50% of BuChE activity, the results are expressed as mean ± SD of three experiments. The results are shown in table 1, in which donepezil is the control experiment result.

Table 1 test for AChE and BuChE inhibition by Compounds

As can be seen from table 1, all the synthesized compounds had some inhibitory effect on BuChE. Among them, compound f9 showed the most prominent inhibitory effect on kinases, IC₅₀1.21 ± 0.19 μ M. The compounds can selectively inhibit the activity of BuChE, and have the potential of developing drugs for treating AD.

Example 23: neuroprotective study

At H₂O₂Neuroprotective effects of compounds were assessed by testing their effect on cell survival in an induced PC12 cell model.

PC12 cells were cultured in modified DMEM medium (10% fetal bovine serum, penicillin 100U/mL, streptomycin 100U/mL). Cell viability was determined by the MTT method. PC12 cell (4X 10)⁴Per well) were seeded in 96-well plates. With 5% CO at 37 deg.C₂After 24 hours of incubation, 100 μ L of test compound was added to each well at different concentrations (30 and 50 μ M). mu.L of MTT solution (5mg/mL) was added to each well under dark conditions, followed by incubation for 4 hours and absorbance was measured at 570nm using a multifunctional microplate reader.

PC12 cells (4X 10)⁴One well) was inoculated in a 96-well plate and cultured for 24h (37 ℃, 5% CO)₂). Discarding old media, adding pre-prepared media containing different concentrations of test compound f1-f21 for pretreatmentFor 3 hours, curcumin was used as a positive control. Exposing cells to H₂O₂Absorbance was measured after 24 hours (250 μ M) using MTT method.

Cell viability was obtained by the MTT method and the results are expressed as mean ± SD of three experiments and are shown in table 2:

table 2 test compounds vs H₂O₂Induced protection of PC12 cells

As can be seen from Table 2, test compounds f1-f21 increased H₂O₂Induced survival of PC12 cells, with compound f9 showing the best inhibitory activity. Compound f9 at 20. mu.M, H₂O₂The survival rate of the induced PC12 cells is 66.4 +/-1.8 percent and is obviously better than that of the positive medicine zingiberin. This indicates that the compounds have some neuroprotective effect.

Example 24: morris Water maze test

1. Experimental materials: male C57BL mice (18-23g), 8 weeks old, were purchased from the university of medicine laboratory center, Anhui. The mice are raised under standard conditions and are kept in an environment with controllable temperature and humidity (23-25 ℃, 40-60 percent and 12 hours).

2. Treatment and modeling:

30 mice were randomly divided into 5 groups, and the normal group and the model group were gavaged with a 0.5% sodium carboxymethylcellulose (CMC-Na) solution for 7 days, and the drug group and the positive control group were gavaged with different concentrations of compound f9(30mg/kg, 10mg/kg) and Donepezil (10mg/kg), respectively. On day 7, except for the normal group (normal saline), the other groups of mice were injected with scopolamine to establish an AD model.

Morris Water maze test

The Morris Water Maze (MWM) device mainly comprises a circular water pool (with the diameter of 150 cm and the height of 60 cm), an escape platform with the diameter of 10 cm, a data acquisition system and an analysis system. The round basin was first filled with water (1.0 cm above the escape platform) and titanium dioxide (0.25g/L) was added to make the water cloudy. The platform was fixed in the third quadrant (four total quadrants) of the round pool and all mice were trained for 4 days.