A selective inhibitor of butyrylcholinesterase and methods of making and using the same

Abstract

The application belongs to the field of medicine, and relates to a butyrylcholinesterase selective inhibitor and a preparation method and application thereof. The application provides a brand-new butyrylcholinesterase selective inhibitor skeleton, in-vitro verification shows that part of the compounds have extremely high butyrylcholinesterase inhibitory activity and selectivity, in-vivo testing finds that a representative compound has an obvious cognitive improvement effect on an AD mouse model, and the butyrylcholinesterase selective inhibitor provided by the application has the potential to be used as a drug for preventing or treating Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to a selective inhibitor of butyrylcholinesterase, its preparation method, and its uses. Background Technology

[0002] Alzheimer's disease (AD) is one of the most common and deadliest neurodegenerative diseases in the world. It is familial and lifestyle-related, affecting over 47 million people globally, and its incidence is projected to exceed 130 million by 2050. More than 50 million people worldwide are believed to have dementia, and AD accounts for over 90% of all dementia cases globally. AD typically includes behavioral and psychological problems. The disease usually affects people over 60. However, recent research indicates that pathological changes in AD begin as early as 15 years before clinical symptoms appear, and by the time a diagnosis is confirmed, irreversible damage to the brain system has usually already occurred. Memory loss and language impairment are signs of damage to nerve cells involved in thinking, memory, and learning. As a result, patients become unable to perform routine tasks, gradually become bedridden, and ultimately die. Furthermore, epidemiological studies suggest that mild cognitive impairment (MCI) is often considered a precursor to AD. Compared to the 1-2% AD conversion rate in healthy aging, 10-15% of MCI patients develop AD each year. Early-onset Alzheimer's disease (AD) is estimated to account for 5% to 10% of AD cases, with a higher reported frequency recently. Furthermore, the increasing prevalence of AD cases in middle- to middle-income countries underscores the urgency for more effective and cost-efficient treatments and for improving the quality of life for patients and their families.

[0003] Currently, the etiology and pathogenesis of AD remain unclear. Its typical pathological features include neuroinflammatory plaques (NPs, also known as age spots (SPs)) formed by β-amyloid (Aβ) deposition, neurofibrillary tangles (NFTs) formed by abnormal tau protein aggregation, and granular vacuolation due to neuronal loss and axonal synaptic abnormalities. Because the exact etiology and pathogenesis of AD are not yet elucidated, its formation is believed to be caused by the combined effects of multiple factors. Current hypotheses regarding its pathogenesis include the cholinergic hypothesis, the Aβ cascade hypothesis, the abnormal phosphorylation hypothesis of tau protein, the neuroinflammatory hypothesis, the oxidative stress hypothesis, and the metal ion disorder hypothesis. Studies show that the onset and progression of AD often involve the cholinergic system, with cholinesterase (ChE) playing a crucial role.

[0004] Although several potential strategies for treating Alzheimer's disease (AD) have been proposed, they are all still in the basic research stage. Currently, the only clinically effective drugs, besides amimantan (an N-methyl-D-aspartic acid receptor (NMDAR) blocker), are cholinesterase inhibitors (donepezil, galantamine, and rivastigmine). These marketed drugs are only suitable for symptomatic treatment of mild to moderate AD; there is a severe lack of drugs effective for severe AD. Furthermore, the efficacy of these drugs is limited, and they also exhibit various dose-related side effects, especially at higher doses. Galantamine and donepezil are acetylcholinesterase (AChE) inhibitors; compared to butyrylcholinesterase (BChE), donepezil has a high selectivity for AChE. The AChE inhibitory activities (IC50 values) of tacrine, donepezil, rivastigmine, and physostigmine are 77, 6.7, 4.3, and 0.67 nM, respectively. Currently, the only effective drug for severe Alzheimer's disease is imimanta, but its efficacy is not entirely satisfactory. Alzheimer's disease has a long course, and many patients with severe Alzheimer's remain without available treatments.

[0005] Drug development based on the cholinergic neuron hypothesis is an important direction in the treatment of Alzheimer's disease (AD), and most commercially available drugs (donepezil, galantamine, rivastigmine, etc.) belong to this category. This hypothesis posits that acetylcholine (ACh) dysfunction is closely related to the typical pathological features of AD: cognitive impairment, decreased learning ability, and memory loss. Simultaneously, ACh can be degraded by cholinesterases (ChEs), which are mainly composed of AChE and BChE. The idea of ​​ACh deficiency as the pathophysiological basis of AD was first proposed in 1976, based on observations of choline acetyltransferase (ChAT), responsible for ACh synthesis, in the post-mortem brains of AD patients. The findings revealed a significant reduction in ChAT in the amygdala, cortex, and hippocampus of post-mortem AD patients compared to non-AD patients. Cholinergic neurons, particularly those located in the basal forebrain, play a crucial role in memory, attention, and learning; the degeneration of ACh-producing neurons in AD patients affects neuronal communication, leading to memory impairment. According to immunoprecipitation data, AChE is an enzyme responsible for degrading ACh, which binds to and interacts with PS-1 in the same cell of neurons in the central nervous system.

[0006] Currently, the first and primary class of drugs used for Alzheimer's disease (AD) are ChE inhibitors. These inhibitors have different specificities for AChE and BChE and are widely used for symptomatic treatment of mild to moderate AD. In the brain, ACh is degraded by AChE and BChE. AChE is present in neural synaptic connections, while BChE is present in glial cells. In a healthy brain, AChE activity is the primary enzyme responsible for breaking down ACh, while in the brains of AD patients, AChE activity is reduced, and BChE activity is increased to compensate for the reduced AChE activity. Four AChE inhibitors have been approved for the treatment of AD, exhibiting different specificities for AChE and BChE. Tacrine was the first drug approved in 1993; however, it was withdrawn in 2013 due to frequent reports of elevated liver enzymes and fatal liver toxicity. The remaining three AChE inhibitors are donepezil, rivastatin, and galantamine. In addition to its role as an AChE inhibitor, galantamine also acts as a positive allosteric modulator of nicotinic acetylcholine receptors and enhances cholinergic neurotransmission. All currently available AChE inhibitors are affected by the same common gastrointestinal side effects (diarrhea and vomiting), which is related to their systemic effects on cholinergic transmission, although donepezil has been reported to have fewer side effects. Donepezil is the most widely used existing AChE inhibitor for mild, moderate, and severe AD, and is also used in combination with the N-methyl-D-aspartate receptor (NMDAR) modulator memantine.

[0007] Compared to AChE inhibitors, BChE inhibitors play a more significant role in Alzheimer's disease (AD). Studies show that as AD progresses from mild to severe, AChE activity in the cerebral cortex gradually decreases, falling by 10%–15% compared to normal brain tissue, while BChE activity gradually increases, reaching up to 120%. This indicates that with the progression of AD, the absolute and relative levels of BChE increase, its activity intensifies, and its expression site expands from white matter to the cortex, suggesting a potential link between BChE content, activity, and expression site and cognitive function.

[0008] Multiple studies have shown that BChE plays a crucial pathophysiological role in the pathogenesis and progression of Alzheimer's disease (AD). On one hand, in normal brain tissue, BChE is expressed in glial cells, primarily distributed in the subcortical region. During AD progression, glial cells proliferate and migrate, extending from the subcortical to the cortical region. The absolute and relative amounts of BChE secreted by these cells increase, along with their activity. This expansion of expression to the cortex leads to increased ACh breakdown and downregulation, resulting in impaired cholinergic neurotransmitter transmission and contributing to or exacerbating AD. On the other hand, the interaction between BChE and Aβ contributes to AD pathogenesis and progression. BChE appears in the pre-NP formation stage, mediating Aβ deposition and increasing NP toxicity, playing a vital role in Aβ pathogenesis. Conversely, Aβ can damage synaptic function of cholinergic neurons, and the proliferation of glial cells around NPs and increased BChE activity further promote AD pathogenesis and progression.

[0009] In conclusion, specific inhibition of BChE is highly rational and has significant research and application value for the treatment of Alzheimer's disease (AD). However, most existing BChE inhibitors emerged alongside AChE inhibitors, and they suffer from drawbacks such as limited quantity, lack of structural novelty and diversity, and poor selectivity. Therefore, developing highly selective BChE inhibitors with novel scaffolds is of great significance and value. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention provides a selective BChE inhibitor, its preparation method, and its uses.

[0011] The technical solution of the present invention is as follows: The first aspect of the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof.

[0012]

[0013] R is selected from substituted or unsubstituted aryl or heteroaryl groups.

[0014] In some implementations, R is selected from quinolinyl or isoquinolinyl, wherein The quinolinyl or isoquinolinyl group may optionally be substituted with one or more R1 groups;

[0015] R1 is selected from halogen, C1-C3 alkyl, nitro, C1-C3 alkoxy, or amino.

[0016] In some implementations, R is selected from , , , , , , , , , , , , , , , , , , , , or .

[0017] In some implementations, R is selected from , , , , , , , , , , , , , or .

[0018] In some embodiments, the present invention provides compounds or pharmaceutically acceptable salts thereof,

[0019] .

[0020] In some typical embodiments, the present invention provides the following compounds or pharmaceutically acceptable salts thereof,

[0021] .

[0022] Secondly, the present invention provides a method for preparing the compound shown in Formula I, comprising the following steps:

[0023]

[0024] 1) In a first solvent, ammonium thiocyanate and compound i are reacted to obtain compound ii;

[0025] 2) In a second solvent, the compound of formula ii and the compound of formula ii-1 are reacted to obtain the compound of formula iii;

[0026] 3) In a third solvent, in the presence of basic substance A and a condensing agent, the compound of formula iii and the compound of formula iii-1 are reacted to obtain the compound of formula I;

[0027] The first solvent, the second solvent, and the third solvent are each independently selected from one or more of water, toluene, xylene, dichloromethane, dichloroethane, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, and acetone;

[0028] Alkaline substance A is selected from one or more of the following: triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, sodium carbonate, sodium bicarbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium hydride, and sodium amino acid.

[0029] The condensing agent is selected from EDC, DIC, DCC, EDCI, DMAP, HOBt, or HOSu.

[0030] In some implementations, the reaction conditions in step 1) are 50-60°C for 1-2 hours.

[0031] In some implementations, the reaction conditions in step 2) are 20-25°C for 10-12 hours.

[0032] In some implementations, the reaction conditions in step 3) are 20-25°C for 12-18 hours.

[0033] Thirdly, the use of the compound of formula I provided by the present invention in the preparation of products for inhibiting cholinesterase activity.

[0034] In some embodiments, the compound of formula I provided by the present invention is used in the preparation of a medicament for selectively inhibiting butyrylcholinesterase activity.

[0035] In some typical embodiments, the compound of formula I provided by the present invention is used in the preparation of drugs for the prevention or treatment of AD.

[0036] Beneficial effects of the present invention: The present invention provides a series of compounds represented by formula (I), and evaluates the efficacy of compounds represented by formula (I) in treating AD (especially moderate to severe AD) by screening for BChE inhibitory activity and selectivity and by Morris water maze test. It was found that they have good in vitro and in vivo activity and extremely high selectivity, and can be used as precursors for further development to exert AD effects by selectively inhibiting BChE. Attached Figure Description

[0037] Figure 1 Time to plateau in mice;

[0038] Figure 2 The trajectory of mice as they reached the platform (A: control group; B: sham-operated group; C: model group; D: tacrine group; E: compound 1 treatment group; F: compound 16 treatment group). Detailed Implementation

[0039] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the invention to the scope of the embodiments described.

[0040] Example 1

[0041] (1) Synthesis of 2-fluorobenzoyl isothiocyanate (intermediate 1)

[0042] Ammonium thiocyanate (500 mg, 6.57 mmol) was placed in a flask, dissolved in acetone (3 ml), and stirred at room temperature. 2-Fluorobenzoyl chloride (624.92 mg, 3.94 mmol) was dissolved in acetone (1 ml) and added dropwise to the flask. The resulting mixture was heated to 58 °C and refluxed for 1 hour to give intermediate 1 (2-fluorobenzoyl isothiocyanate).

[0043] (2) Synthesis of N-(2-ethylphenyl)-3-(2-fluorophenyl)-3-oxopropylthioamide (intermediate 2)

[0044] The reaction mixture of intermediate 1 in the conical flask was cooled to room temperature, and then 2-ethylaniline (396 mg, 3.27 mmol) was added. The mixture was stirred at room temperature for 12 hours. Water and ethanol were added to the reaction system, followed by multiple extractions with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After drying, the organic phase was purified by silica gel column chromatography using petroleum ether and ethyl acetate as eluents to give intermediate 2 (N-(2-ethylphenyl)-3-(2-fluorophenyl)-3-oxopropylthioamide).

[0045] (3) Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(2-ethylphenyl)uryl)-2-fluorobenzamide:

[0046] Intermediate 2 (489 mg, 1.62 mmol) was dissolved in 5 mL of dichloromethane in a pear-shaped flask. Triethylamine (246 mg, 2.43 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (465 mg, 2.43 mmol), and tryptophan (386 mg, 2.43 mmol) were then added sequentially. After reacting at room temperature for 16 hours, water was added to the reaction mixture, followed by multiple extractions with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After drying, the organic phase was separated by silica gel column chromatography using petroleum ether and ethyl acetate as eluents to give a pale yellow solid compound W1 with a purity of 95.6% and a yield of 75%. Thin-layer chromatography (TLC) analysis showed a single spot that appeared dark under 254 nm UV light and showed no fluorescence under 365 nm UV light.1 H NMR (600 MHz, CDCl3) δ 11.80 (s, 1H), 8.21 (s, 1H), 8.16 (t, J = 7.1 Hz, 1H), 7.56 (d, J = 7.7 Hz, 1H), 7.41 (q, J = 6.6 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.25 - 7.24 (m, 1H), 7.19 - 7.16 (m, 3H), 7.13 -7.09 (m, 1H), 7.07 (t, J = 7.0 Hz, 2H), 6.96 (d, J = 7.6 Hz, 1H), 6.89 (s,1H), 3.79 (q, J = 6.5 Hz, 2H), 3.03 (t, J = 6.2 Hz, 2H), 2.58 (q, J = 7.6 Hz, 2H), 2.05 (s, 1H), 1.13 (t, J = 7.6 Hz, 3H). 13 C NMR (150 MHz, DMSO-d6) δ175.1, 160.4, 159.3, 140.1, 136.8, 134.4, 132.5, 132.0, 130.0, 128.5, 127.8,127.7, 127.2, 124.2, 123.2, 121.5, 119.0, 118.7, 117.1, 116.9, 112.2, 111.9,42.3, 26.1, 24.7, 25.0. HR-MS (ESI): calcd. for C 26 H 26 FN4O [M+H + 429.2085, found 429.2115.

[0047] Example 2

[0048] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(3-chlorophenyl)uryl)-2-fluorobenzamide:

[0049] Following the synthesis method of Example 1, replacing 2-ethylaniline with 3-chloroaniline yielded a pale yellow solid compound W2 (purity 95.2%) in 62% yield. Thin-layer chromatography analysis showed a single spot with a dark spot under 254 nm UV light and no fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 12.00 (s, 1H), 8.38 (s, 1H), 8.04 (d, J =89.7 Hz, 1H), 7.61 (d, J = 27.2 Hz, 1H), 7.47 - 7.39 (m, 1H), 7.33 (d, J =8.2 Hz, 1H), 7.19 (t, J = 7.4 Hz, 2H), 7.14 - 6.91 (m, 6H), 6.79 (d, J = 35.8Hz, 1H), 3.80 (s, 2H), 3.09 (s, 2H), 2.04 (s, 1H). 13 C NMR (150 MHz, DMSO-d6)δ 174.8, 161.9, 160.3, 158.4, 136.9, 136.9, 132.6, 132.6, 131.7, 128.2,128.2, 127.6, 124.2, 123.8, 121.6, 118.9, 118.9, 118.8, 117.1, 116.9, 112.0,112.0, 42.5, 25.3. HR-MS (ESI): calcd. for C 24 H 21 ClFN4O [M+H + ] 435.1388 found 435.1430.

[0050] Example 3

[0051] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(2-methoxyphenyl)uryl)-2-fluorobenzamide:

[0052] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 2-methoxyaniline to obtain W3 (purity 95.2%), a light yellow solid compound with a yield of 64%. Thin-layer chromatography showed a single spot, appearing as a dark spot under 254 nm UV light and showing no fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 11.75 (s, 1H), 8.39 (s, 1H), 8.15 (s, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.44 - 7.38 (m, 1H), 7.33 (d, J = 8.1Hz, 1H), 7.19 - 7.12 (m, 2H), 7.15 - 7.06 (m, 3H), 6.95 (s, 2H), 6.87 - 6.85(m, 1H), 6.79 (s, 1H), 3.81 (s, 2H), 3.70 (s, 3H), 3.06 (s, 2H), 2.05 (s,1H). 13 C NMR (150 MHz, DMSO-d6) δ 174.7, 162.0, 160.3, 158.4, 152.9, 136.8,132.4, 131.4, 131.9, 128.6, 127.8, 127.5, 125.5, 124.2, 123.2, 121.5, 119.0,118.7, 117.1, 116.9, 112.7, 111.9, 56.2, 42.3, 26.0. HR-MS (ESI): calcd. forC 25 H 24 FN4O2[M+H + ] 431.1883 found 431.1902..

[0053] Example 4

[0054] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(3-nitrophenyl)ureyl)-2-fluorobenzamide:

[0055] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 3-nitroaniline to obtain a yellow solid compound W4 (purity 96.2%) in 74% yield. Thin-layer chromatography showed a single spot, appearing as a dark spot under 254 nm UV light and without fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 8.43 (s, 1H), 8.14 (s, 1H), 7.99 (s, 1H), 7.88 (s, 1H), 7.74 (s, 1H), 7.68 (s, 1H), 7.46 - 7.38 (m, 3H), 7.23 - 7.15(m, 5H), 7.00 (s, 1H), 3.78 (s, 2H), 3.16 (s, 2H), 2.04 (s, 1H). 13 C NMR (150MHz, DMSO-d6) δ 175.0, 162.0, 160.3, 158.4, 148.1, 140.3, 136.9, 132.7,129.7, 127.9, 127.5, 124.3, 123.8, 121.6, 118.9, 118.9, 118.5, 118.1, 117.1,116.9, 112.0, 111.3, 42.6, 25.3. HR-MS (ESI): calculated for C 24 H 21 FN5O3 [M+H + 446.1628 found 446.1653.

[0056] Example 5

[0057] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(m-tolyl)aminoacyl)-2-fluorobenzamide:

[0058] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 3-methylaniline to obtain a light brown solid, W5 (purity 96.2%), in a yield of 75%. Thin-layer chromatography analysis showed only one spot, which appeared as a dark spot under 254 nm UV light and showed no fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 11.88 (s, 1H), 8.12 (s,2H), 7.59 (d, J = 7.4 Hz, 1H), 7.40 (q, J = 6.9 Hz, 1H), 7.35 (d, J = 8.1 Hz,1H), 7.20 - 7.06 (m, 5H), 6.98 (d, J = 11.5 Hz, 2H), 6.82 (d, J = 6.7 Hz, 2H), 3.81 (s, 2H), 3.07 (s, 2H), 2.22 (s, 3H), 2.04 (s, 1H). 13 C NMR (150 MHz, DMSO-d6) δ 174.7, 162.0, 160.3, 158.6, 136.9, 136.9, 132.5, 132.4, 131.8,131.8, 128.5, 128.4, 125.3, 124.3, 121.8, 121.5, 119.0, 118.8, 117.0, 116.9,111.9, 111.9, 42.4, 25.5, 21.5. HR-MS (ESI): calcd. for C 25 H 24 FN4O [M+H + ]415.1934, found 415.1970.

[0059] Example 6

[0060] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(4-chlorophenyl)uryl)-2-fluorobenzamide:

[0061] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 4-chloroaniline to obtain W6 (purity 97.8%) as a white solid with a yield of 73%. Thin-layer chromatography (TLC) analysis showed a single spot, appearing as a dark spot under 254 nm UV light and without fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 11.92 (s, 1H), 8.22 (s, 1H), 8.12 (s,2H), 7.62 (d, J = 61.1 Hz, 1H), 7.42 (s, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.23- 7.11 (m, 6H), 6.95 (s, 1H), 6.84 (s, 2H), 3.80 (s, 2H), 3.06 (s, 2H). 13 CNMR (150 MHz, DMSO-d6) δ 174.8, 161.9, 160.3, 158.8, 136.8, 136.8, 132.5,132.5, 131.8, 128.3, 128.2, 127.6, 124.3, 124.3, 123.8, 121.6, 118.9, 118.9,118.8, 117.0, 116.9, 112.0, 42.5, 25.2. HR-MS (ESI): calcd. for C 24 H 21 ClFN4O [M+H + ] 435.1388, found 435.1443.

[0062] Example 7

[0063] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(4-methoxyphenyl)uryl)-2-fluorobenzamide:

[0064] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 4-methoxyaniline to obtain W7 (purity 96.2%), a white solid with a yield of 69%. Thin-layer chromatography analysis showed a single spot, which appeared as a dark spot under 254 nm UV light and showed no fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 11.68 (s, 1H), 8.18 (s, 1H), 8.12(s, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.42 - 7.38 (m, 1H), 7.34 (d, J = 8.1 Hz,1H), 7.20 - 7.15 (m, 2H), 7.12 - 7.06 (m, 2H), 6.94 - 6.92 (m, 3H), 6.75 (d,J = 8.7 Hz, 2H), 3.81 - 3.77 (m, 2H), 3.76 (s, 3H), 3.05 - 3.03 (m, 2H). 13 CNMR (150 MHz, DMSO-d6) δ 174.8, 161.9, 160.2, 159.2, 136.8, 132.3, 128.7,128.6, 127.5, 124.2, 124.2, 123.3, 121.5, 119.0, 118.7, 117.0, 116.8, 115.4,113.7, 111.9, 100.0, 55.8, 42.3, 25.9. HR-MS (ESI): calcd. for C 25 H 24 ClFN4O2 [M+H + ] 431.1883, found 431.4043.

[0065] Example 8

[0066] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(2-chlorophenyl)uryl)-2-fluorobenzamide:

[0067] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 2-chloroaniline to obtain W4 (95.0% purity) as a white solid, with a yield of 64%. Thin-layer chromatography analysis showed a single spot, dark under 254 nm UV light and without fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 12.07 (s, 1H), 8.38 (s, 1H), 8.20 (s, 1H), 7.99 (s, 1H), 7.69 - 7.59 (m, 1H), 7.43 (s, 2H), 7.33 (d, J = 8.1 Hz, 1H),7.20-7.17 (m, 2H), 7.11 - 7.03 (m, 5H), 3.82 (s, 2H), 3.22 (s, 2H), 2.05 (s,1H). 13 C NMR (150 MHz, DMSO-d6) δ 175.0, 162.1, 160.4, 158.3, 136.8, 134.2,132.7, 131.9, 130.7, 128.9, 128.1, 127.8, 127.4, 124.3, 123.3, 121.5, 119.0,118.7, 117.1, 117.0, 112.2, 111.9, 42.2, 25.9. HR-MS (ESI): calcd. forC 24 H 21 ClFN4O [M+H + ] 435.1388, found 435.1422.

[0068] Example 9

[0069] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(3-methoxyphenyl)uryl)-2-fluorobenzamide:

[0070] Following the synthesis method of Example 1, replacing 2-ethylaniline with 3-methoxyaniline yielded a white solid, W9 (97.0% purity), in a yield of 67%. Thin-layer chromatography analysis showed it as a single spot, appearing as a dark spot under 254 nm UV light and showing no fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 11.95 (s, 1H), 8.11 (s, 1H),8.06 (s, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.40 (s, 1H), 7.35 (d, J = 8.6 Hz,1H), 7.26 - 7.25 (m, 1H), 7.20 - 7.08 (m, 5H), 6.98 (s, 1H), 6.73 (s, 1H), 6.60 (s, 1H), 3.81 (s, 1H), 3.67 (s, 3H), 3.07 (s, 2H), 2.04 (s, 1H). 13 C NMR(150 MHz, DMSO-d6) δ 174.8, 161.9, 160.3, 158.5, 136.8, 132.5, 131.8, 130.9,129.3, 128.5, 127.7, 124.2, 123.3, 121.5, 119.0, 118.8, 117.0, 116.9, 116.5,112.4, 111.9, 109.9, 55.6, 42.4, 25.7. HR-MS (ESI): calcd. for C 25 H 24 FN4O2 [M+H + ] 431.1883, found 431.4014.

[0071] Example 10

[0072] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(o-tolyl)ureyl)-2-fluorobenzamide:

[0073] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 2-methylaniline to obtain W10 (purity 98.6%), a pale white solid with a yield of 69%. Thin-layer chromatography analysis showed a single spot, which appeared as a dark spot under 254 nm UV light and showed no fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 11.77 (s, 1H), 8.24 (s, 1H), 8.17 (t, J =7.7 Hz, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.41 (q, J = 6.9 Hz, 1H), 7.33 (d, J =8.1 Hz, 1H), 7.24 - 7.04 (m, 7H), 6.97 (d, J = 7.8 Hz, 1H), 6.90 (s, 1H), 3.80 (t, J = 6.5 Hz, 2H), 3.04 (t, J = 6.8 Hz, 2H), 2.21 (s, 3H), 2.05 (s,1H). 13 C NMR (150 MHz, DMSO-d6) δ 174.9, 162.1, 160.4, 159.0, 136.8, 135.1,134.2, 132.5, 131.9, 131.6, 128.5, 127.8, 127.7, 127.4, 126.7, 124.2, 123.2,121.5, 119.0, 118.7, 117.1, 116.9, 112.2, 111.9, 42.3, 26.1, 18.1. HR-MS(ESI): calcd. for C 25 H 24 FN4O [M+H + ] 415.1934, found 415.4065.

[0074] Example 11

[0075] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(p-tolyl)ureyl)-2-fluorobenzamide:

[0076] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 4-methylaniline to obtain a pale yellow solid, W11 (purity 97.4%), in a yield of 51%. Thin-layer chromatography analysis showed a single spot, which appeared as a dark spot under 254 nm UV light and showed no fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 11.81 (s, 1H), 8.09 (s,2H), 7.57 (d, J = 7.9 Hz, 1H), 7.41-7.35 (m, 2H), 7.21 - 7.15 (m, 2H), 7.12 -7.05 (m, 4H), 6.97 (s, 1H), 6.90 (d, J = 6.7 Hz, 2H), 3.78 (s, 2H), 3.05 (s,2H), 2.30 (s, 3H), 2.04 (s, 1H). 13 C NMR (150 MHz, DMSO-d6) δ 174.8, 161.9,160.3, 158.8, 136.8, 136.8, 132.3, 131.8, 130.6, 128.6, 128.5, 127.7, 125.2,124.2, 121.5, 119.0, 119.0, 118.8, 117.0, 116.9, 111.9, 111.9, 42.4, 25.9,21.0. HR-MS (ESI): calcd. for C 25 H 24 FN4O [M+H + ] 415.1934, found 415.4071.

[0077] Example 12

[0078] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(2-nitrophenyl)ureyl)-2-fluorobenzamide:

[0079] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 2-nitroaniline to obtain W12 (purity 95.2%), a yellow solid with a yield of 55%. Thin-layer chromatography analysis showed a single spot, which appeared as a dark spot under 254 nm UV light and showed no fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 8.34 (s, 1H), 8.06 (s, 1H), 7.97 (s, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.55 (s, 1H), 7.45 (s, 1H), 7.36 (d,J = 7.8 Hz, 1H), 7.25 - 7.10 (m, 5H), 7.03 (s, 1H), 3.77 (s, 2H), 3.20 (s,2H), 2.04 (s, 1H). 13 C NMR (150 MHz, DMSO-d6) δ 177.2, 145.1, 144.1, 143.4,136.8, 135.3, 134.0, 132.9, 132.0, 130.8, 127.8, 126.1, 125.1, 124.3, 123.4,122.0, 121.5, 119.0, 118.8, 116.8, 112.3, 112.0, 42.5, 25.2. HR-MS (ESI):calcd. for C 24 H 21 FN5O3 [M+H + ] 446.1628, found 446.1672.

[0080] Example 13

[0081] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(4-nitrophenyl)ureyl)-2-fluorobenzamide:

[0082] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 4-nitroaniline to obtain W13 (purity 98.2%), a yellow solid with a yield of 70%. Thin-layer chromatography analysis showed a single spot, appearing as a dark spot under 254 nm UV light and without fluorescence at 365 nm. 1 H NMR (600 MHz, CDCl3) δ 8.47 (s, 1H), 8.17 (s, 2H), 8.06 (s, 1H), 7.99 (s, 1H), 7.69 (s, 1H), 7.50 (s, 1H), 7.39 (d, J = 7.6 Hz, 1H),7.25 - 7.00 (m, 4H), 6.92 (s, 2H), 3.78 (s, 2H), 3.16 (s, 2H), 2.04 (s, 1H). 13C NMR (150 MHz, DMSO-d6) δ 175.2, 162.0, 160.3, 158.2, 145.6, 142.8, 136.9,132.9, 131.8, 127.8, 127.5, 125.2, 124.6, 124.4, 123.8, 122.7, 121.6, 118.9,117.1, 117.0, 112.0, 111.3, 42.8, 25.3. HR-MS (ESI): calcd. for C 24 H 21 FN5O3 [M+H+] 446.1628, found 446.1686.

[0083] Example 14

[0084] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(2-fluorophenyl)uryl)-2-fluorobenzamide:

[0085] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 2-fluoroaniline to obtain W14 (purity 95.6%), a pale yellow solid with a yield of 75%. Thin-layer chromatography analysis showed a single spot, appearing as a dark spot under 254 nm UV light and without fluorescence at 365 nm. 1 H NMR (600 MHz, CDCl3) δ 11.93 (s, 1H), 8.17 - 7.99 (m,2H), 7.63 (d, J = 54.0 Hz, 1H), 7.42 (s, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.20- 7.01 (m, 9H), 3.81 (s, 2H), 3.13 (d, J = 64.3 Hz, 2H), 2.04 (s, 1H). 13 C NMR(150 MHz, DMSO-d6) δ 174.9, 162.0, 160.4, 158.7, 136.8, 132.6, 131.9, 129.7,128.4, 128.3, 128.2, 127.9, 125.7, 124.4, 124.3, 124.0, 123.2, 121.5, 119.0,118.7, 116.9, 116.1, 42.5, 25.9. HR-MS (ESI): calcd. for C 24 H 21 F2N4O [M+H+ ]419.1683, found 419.3828.

[0086] Example 15

[0087] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(3-fluorophenyl)uryl)-2-fluorobenzamide:

[0088] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 3-fluoroaniline to obtain W15 (purity 95.0%), a pale yellow solid with a yield of 72%. Thin-layer chromatography analysis showed a single spot, which was dark under 254 nm UV light and showed no fluorescence at 365 nm. 1 H NMR (600 MHz, CDCl3) δ 12.05 (s, 1H), 8.57 (s, 1H), 8.21 (s, 1H), 8.13 - 7.98 (m, 1H), 7.67 - 7.59 (m, 1H), 7.42 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.24 - 7.06 (m, 5H), 6.98 - 6.86 (m, 1H), 6.74 (s, 2H), 3.80 (d, J = 28.2 Hz, 2H), 3.12 (d, J = 29.4 Hz, 2H), 2.05 (s, 1H). 13 C NMR(150 MHz, DMSO-d6) δ 174.8, 161.9, 160.2, 158.4, 136.9, 132.6, 132.5, 131.7,128.3, 128.2, 127.6, 124.3, 123.8, 121.6, 119.4, 118.9, 118.9, 118.8, 117.1,116.9, 112.0, 111.0, 42.5, 25.4. HR-MS (ESI): calcd. for C 24 H 21 F2N4O [M+H + ]419.1683, found 419.1711.

[0089] Example 16

[0090] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(4-fluorophenyl)uryl)-2-fluorobenzamide:

[0091] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 4-fluoroaniline to obtain W16 (purity 95.1%), a pale yellow solid with a yield of 74%. Thin-layer chromatography showed a single spot, which appeared as a dark spot under 254 nm UV light and showed no fluorescence at 365 nm. 1 H NMR (600 MHz, CDCl3) δ 11.82 (s, 1H), 8.28 (s, 1H), 8.12(s, 1H), 7.56 (d, J = 7.7 Hz, 1H), 7.41 (d, J = 6.8 Hz, 1H), 7.34 (d, J = 8.2Hz, 1H), 7.21 - 7.17 (m, 2H), 7.13 - 7.07 (m, 2H), 6.93 - 6.89 (m, 5H), 3.82- 3.79 (m, 2H), 3.11 - 3.05 (m, 2H), 2.05 (s, 1H). 13 C NMR (150 MHz, DMSO-d6)δ 174.8, 161.9, 160.3, 158.9, 136.8, 132.4, 132.4, 131.8, 128.4, 128.4,127.7, 126.5, 124.2, 123.7, 121.6, 119.0, 119.0, 118.8, 117.0, 116.9, 115.1,112.0, 42.4, 25.3. HR-MS (ESI): calcd. for C 24 H 21 F2N4O [M+H + ] 419.1683, found419.3821.

[0092] Example 17

[0093] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(2-bromophenyl)uryl)-2-fluorobenzamide:

[0094] Following the synthesis method of Example 1, replacing 2-ethylaniline with 2-bromoaniline yielded W17 (96.6% purity), a pale yellow solid with a yield of 77%. Thin-layer chromatography showed a single spot, appearing as a dark spot under 254 nm UV light and exhibiting no fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 12.04 (s, 1H), 8.35 (s, 1H), 8.08 (s,1H), 7.99 (s, 1H), 7.70 (s, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.46 (s, 1H), 7.35(d, J = 8.1 Hz, 1H), 7.21-6.92 (m, 7H), 3.82 (s, 2H), 3.24 (s, 2H), 2.04 (s,1H). 13 C NMR (150 MHz, DMSO-d6) δ 175.0, 162.1, 160.4, 158.3, 136.8, 135.7,133.8, 132.7, 132.0, 129.5, 128.5, 128.2, 127.7, 124.3, 123.3, 121.5, 119.9,119.0, 118.7, 117.0, 112.2, 111.9, 42.4, 25.9. HR-MS (ESI): calcd. forC 24 H 21 BrFN4O [M+H + ] 479.0883, found 479.0922.

[0095] Example 18

[0096] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(3-bromophenyl)uryl)-2-fluorobenzamide:

[0097] Following the synthesis method of Example 1, replacing 2-ethylaniline with 3-bromoaniline yielded W18 (99% purity), a yellow solid with a yield of 67%. Thin-layer chromatography showed a single spot, appearing as a dark spot under 254 nm UV light and exhibiting no fluorescence at 365 nm. 1H NMR (600 MHz, DMSO-d6) δ 12.04 (s, 1H), 8.53 (s, 1H), 8.16 (s,1H), 8.05 (d, J = 80.5 Hz, 1H), 7.63 (d, J = 51.5 Hz, 1H), 7.42 (s, 1H), 7.36(d, J = 8.2 Hz, 1H), 7.23 - 6.98 (m, 7H), 6.86 (d, J = 49.2 Hz, 1H), 3.79 (d,J = 33.4 Hz, 2H), 3.11 (d, J = 31.0 Hz, 2H), 2.04 (s, 1H). 13 C NMR (150 MHz, DMSO-d6) δ 174.8, 162.0, 160.3, 158.4, 136.9, 136.9, 132.6, 131.8, 128.2, 128.1, 127.6, 126.7, 124.2, 123.8, 122.6, 121.6, 118.9, 118.9, 117.1, 116.9,112.0, 112.0, 42.5, 25.3. HR-MS (ESI): calcd. for C 24 H 21 BrFN4O [M+H + ] 479.0883, found 479.0912.

[0098] Example 19

[0099] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(4-bromophenyl)uryl)-2-fluorobenzamide:

[0100] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 4-bromoaniline to obtain a pale yellow solid, W19 (purity 97.0%), in a yield of 76%. Thin-layer chromatography showed a single spot, appearing as a dark spot under 254 nm UV light and without fluorescence at 365 nm. 1H NMR (600 MHz, CDCl3) δ 11.92 (s, 1H), 8.50 (s, 1H), 8.12 (s,2H), 7.57 (s, 1H), 7.41 (s, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.32 (s, 1H),7.22(t, J = 7.2 Hz, 1H), 7.18 (s, 1H), 7.10 (s, 2H), 6.97 (s, 1H), 6.80 (s, 2H),3.80 (s, 2H), 3.06 (s, 2H), 2.04 (s, 1H). 13 C NMR (150 MHz, DMSO-d6) δ 174.8,161.9, 160.3, 158.5, 136.9, 136.9, 132.5, 132.5, 131.8, 128.3, 128.2, 127.6,126.4, 124.3, 121.6, 118.9, 118.9, 118.8, 117.0, 116.9, 112.0, 112.0, 42.5,25.3. HR-MS (ESI): calcd. for C 24 H 21 BrFN4O [M+H + ] 479.0883, found 479.0903.

[0101] Example 20

[0102] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(quinolin-3-yl)ureyl)-2-fluorobenzamide:

[0103] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 3-aminoquinoline to obtain W20 (purity 96.8%), a pale yellow solid with a yield of 74%. Thin-layer chromatography showed a single spot, which was dark under 254 nm UV light and showed no fluorescence at 365 nm. 1 H NMR (600 MHz, CDCl3) δ 12.25 (s, 1H), 8.58 (s, 1H), 8.17 (s, 1H), 8.00 (s, 2H), 7.71 - 7.37 (m, 7H), 7.25 - 6.90 (m, 5H), 3.84(s, 2H), 3.20 (s, 2H), 2.04 (s, 1H). 13C NMR (150 MHz, DMSO-d6) δ 175.0,161.9, 160.2, 158.9, 149.1, 144.9, 136.9, 136.9, 132.6, 132.5, 131.7, 129.1,128.2, 127.5, 127.4, 124.3, 123.9, 121.6, 119.0, 119.0, 118.9, 117.1, 116.9,112.0, 111.3, 42.6, 25.4. HR-MS (ESI): calcd. for C 27 H 23 FN5O [M+H + ] 452.1887, found 452.4071.

[0104] Example 21

[0105] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(quinolin-6-yl)ureyl)-2-fluorobenzamide:

[0106] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 6-aminoquinoline to obtain W21 (purity 97.9%), a yellow to brown solid with a yield of 79%. Thin-layer chromatography showed a single spot, appearing as a dark spot under 254 nm UV light and without fluorescence at 365 nm. 1 H NMR (600 MHz, CDCl3) δ 12.20 (s, 1H), 8.86 (s, 1H), 8.13 -8.10 (m, 1H), 7.97 (s, 1H), 7.81 - 7.58 (m, 2H), 7.42 - 7.28 (m, 6H), 7.20 -6.96 (m, 5H), 3.85 (s, 2H), 3.12 (s, 2H), 2.03 (s, 1H). 13 C NMR (150 MHz, DMSO-d6) δ 174.8, 162.0, 160.3, 158.7, 136.9, 136.0, 132.6, 131.8, 130.3, 129.9, 128.0, 127.7, 126.5, 126.2, 124.3, 123.7, 122.2, 121.6, 119.0, 119.0,118.9, 117.1, 116.9, 112.0, 112.0, 42.5, 25.5. HR-MS (ESI): calcd. forC27 H 23 FN5O [M+H + ] 452.1887, found 452.1937.

[0107] Example 22

[0108] Synthesis of (E)-N-(N'-(2-(1H-indol-3-yl)ethyl)-N-(quinolin-7-yl)ureyl)-2-fluorobenzamide:

[0109] Following the synthesis method of Example 1, 2-ethylaniline was replaced with 7-aminoquinoline to obtain W22 (purity 95.0%), a light brown solid with a yield of 52%. Thin-layer chromatography showed a single spot, appearing as a dark spot under 254 nm UV light and without fluorescence at 365 nm. 1 H NMR (600 MHz, CDCl3) δ 12.34 (s, 1H), 8.85 (s, 1H), 8.32 (s, 1H), 8.15 (s, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.79 (s, 1H), 7.70 (s,1H), 7.55 (s, 1H), 7.42 - 7.29 (m, 3H), 7.20 - 7.00 (m, 6H), 3.84 (s, 2H), 3.10 (s, 2H), 2.04 (s, 1H). 13 C NMR (150 MHz, DMSO-d6) δ 174.9, 162.0, 160.3,158.6, 151.3, 148.7, 136.9, 136.1, 132.6, 131.8, 128.3, 128.3, 127.6, 124.8,124.3, 123.7, 121.6, 121.0, 119.0, 119.0, 118.9, 117.1, 116.9, 112.0, 112.0,42.6, 25.4. HR-MS (ESI): calcd. for C 27 H 23 FN5O [M+H + ] 452.1887, found 452.4095.

[0110] The structural formulas of the compounds synthesized in Examples 1-22

[0111]

[0112]

[0113]

[0114] The following are the pharmacodynamic tests and results of some compounds in this invention:

[0115] Determination of cholinesterase inhibitory activity:

[0116] Drugs and reagents: The test compound, AChE (EC3.1.1.7, Type VI-S, selected from electric eel), BChE (EC3.1.1.8, selected from horse serum), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), acetylthiocholine (ATC) iodide and butyrylthiocholine (BTC) iodide were purchased from Sigma-Aldrich; the positive control tacrine hydrochloride (9-Amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate) was purchased from BioTrend.

[0117] Instrument: THERMO Varioskan Flash full-wavelength multi-functional microplate reader.

[0118] Experimental methods:

[0119] (1) Preparation of buffer solution: First, dissolve 6.8 g potassium dihydrogen phosphate in 500 mL of ultrapure water and adjust the pH to 8.0 ± 0.1 with potassium hydroxide to obtain phosphate buffered saline (PBS), which is stored at 4 °C.

[0120] (2) Preparation of 0.01 M DTNB solution: Weigh 0.198 g of DTNB solid, dissolve it in 50 mL of ultrapure water, and then add 0.075 g of sodium bicarbonate solid to the solution to obtain 0.01 mol / L DTNB stock solution, which is stored at -20 °C.

[0121] (3) Prepare 0.075 M ATC / BTC solution: Dissolve 0.109 g ATC / 0.119 g BTC solid (0.217 g ATC / 0.237 g BTC solid) in 10 mL of ultrapure water to obtain 0.075 mol / L ATC / BTC stock solution and store it at -20 °C.

[0122] (4) Preparation of AChE and BChE solutions: Dissolve 500 units of AChE / BChE solid in 1 mL of 1% gel aqueous solution, then dilute with ultrapure water to 100 mL to obtain a 5 unit / mL AChE / BChE stock solution, and store at -20 °C.

[0123] (5) Preparation of test solution: Dissolve the solid test compound in DMSO to obtain 10 -1 The compound stock solution was stored at 4 °C. During testing, it was serially diluted with methanol to obtain concentrations of 10 mol / L. -4 mol / L, 10 -5 mol / L, 10 -6 mol / L, 10 -7 mol / L, 10 -8 mol / L, 10 -9 A mol / L compound solution.

[0124] Before the experiment, remove all solutions stored at low temperature and allow them to dissolve completely at room temperature. Take an appropriate amount of AChE / BChE stock solution and dilute with ultrapure water to obtain an enzyme solution of 0.75 units / mL. Take an appropriate amount of DTNB stock solution and dilute with ultrapure water to obtain a DTNB solution of 0.0015 mol / L. Take an appropriate amount of ATC / BTC stock solution and dilute with ultrapure water to obtain a DTNB solution of 0.0023 mol / L. At 25 °C, add 40 µL of PBS, 10 µL of the compound solution, 10 µL of AChE / BChE solution, 20 µL of DTNB solution, and 20 µL of ATC / BTC solution sequentially to a 96-well plate. Seven test groups were set up according to different compound concentrations, with three replicates per group: 0 mol / L, 10 µL of DTNB, 10 µL of AChE / BChE solution, 10 µL of DTNB, 10 µL of ATC / BTC solution, and 10 µL of ATC / BTC solution to each well. -4 mol / L, 10 -5 mol / L, 10 -6 mol / L, 10 -7 mol / L, 10 -8 mol / L, 10 -9The concentration was mol / L. The 0 mol / L test group served as the negative control group, using an equal volume of chromatographic methanol instead of the compound solution. Additionally, since the added DTNB solution has a certain color and UV absorption, a blank control group was required. In this group, only 80 µL of PBS and 20 µL of DTNB solution were added to each of the three replicates to eliminate the influence of DTNB on the experimental results. The reaction proceeded rapidly after adding ATC / BTC solution to the test group. The 96-well plate was incubated at 37°C for 5 minutes, and then the UV absorbance of each group was measured at 405 nm (412 nm on paper). The UV absorbance of the negative control group was defined as 100%, and Experiment A was defined as the UV absorbance of the test group, while Blank A was defined as the UV absorbance of the blank control group. The inhibition rate of each concentration of compound in the test group was calculated using the following formula: IR(%) = (1 - Experiment A / Blank A) * 100%. The inhibition rates calculated at each compound concentration were used to perform nonlinear regression analysis using GraphPad Prism 8.0™ (GraphPadSoftware, San Diego, CA, USA) software to obtain the half-maximal inhibition rate (IC50), as shown in Table 1.

[0125]

[0126] Table 1. Test results of each compound on human cholinesterase.

[0127]

[0128]

[0129] a This indicates the inhibition rate of the compound against the target at a concentration of 10 μM.

[0130] The compounds in Table 1 exhibited good inhibitory activity against BChE (e.g., compounds 1, 3, 6, 11, 14, and 16), but low inhibitory activity against AChE (inhibition rate less than 30% at 10 μM concentration), indicating that this series of compounds has extremely high selectivity. In the early stages of Alzheimer's disease (AD), 80% of ACh is hydrolyzed by AChE, with BChE having almost no effect. As the disease progresses, AChE levels decline and its function is almost lost. At this point, BChE levels and function relatively increase, replacing AChE as the main metabolic enzyme for ACh. Therefore, the inhibitory activity of BChE is particularly important for the treatment of severe AD. To prevent compounds from affecting peripheral AChE and causing unnecessary peripheral cholinergic side effects, the development of selective BChE inhibitors is of great significance. The compounds involved in this invention have excellent inhibitory activity and extremely high selectivity against BChE, and are expected to produce good therapeutic effects for mild and severe AD.

[0131] Morris water maze study and mouse behavior research

[0132] Instrument: Panlab SMART 3.0 Behavioral Video Analyzer

[0133] Animals: Male ICR mice (4 weeks old, weighing 18-20 grams) were purchased from the Haoyuan Animal Platform of Qingdao University.

[0134] Reagents: Tacrine (purity >95%), compound 1, compound 16.

[0135] Experimental Methods: Sixty mice were randomly divided into six groups (n=10 per group): control group, sham-operated group, model group, tacrine group, compound 1 treatment group, and compound 16 treatment group. Mice in the sham-operated group received intraventricular injection of saline, while mice in the model group, tacrine group, compound 1 treatment group, and compound 16 treatment group received intraventricular injection of Aβ (2 g / L). The control group received no treatment. On the second day after modeling, tacrine, compound 1, and compound 16 were dissolved in 1% DMSO solution and 99% saline solution, respectively, and administered by gavage (10 mg / kg body weight). Cognitive function and memory ability of the mice were tested using a water maze. An escape platform (10 cm in diameter) was fixed in a circular pool (120 cm in diameter, 60 cm in height), with a 5 cm high flag fixed on the platform. The pool was filled with water to a height of 40 cm, and the temperature was maintained at 25 °C to form a water maze. On days 1-2 after drug administration, mice were placed on an escape platform for training. From days 3-5, the platform was submerged 1 cm underwater for further training. On the final day (day 6), the platform was removed, and the mice were evaluated. The time, trajectory, and speed at which the mice reached the platform were recorded. The experimental results are shown in Table 2. Figure 1 , Figure 2 As shown.

[0136] Table 2 Time it takes for mice to reach the platform location

[0137] Group control group Sham surgery group Model group Taklin Group Compound 1 treatment group Compound 16 treatment group Arrival time (s) 22.51 ± 2.05 <![CDATA[25.06 ± 0.66 #### ]]> <![CDATA[71.52 ± 7.40 **** ]]> <![CDATA[18.27 ± 0.77 **** ]]> <![CDATA[18.82 ± 0.12 **** ]]> <![CDATA[19.99 ± 1.24 **** ]]>

[0138] #### A p-value < 0.0001 indicates a significant difference. ** A p-value < 0.01 indicates a significant difference. *** A p-value < 0.001 indicates a significant difference. **** A p-value < 0.0001 indicates a significant difference.

[0139] Results analysis: Based on Table 2, Figure 1 , Figure 2The results showed that, compared with the control group, the average time for mice in the model group to reach the platform was significantly increased, indicating that Aβ causes memory deficits in mice and that the model was successfully established. Compared with the model group, the time and distance reached in the tacrine group were significantly reduced, indicating that tacrine significantly improves the memory and cognitive functions of mice. The time and distance reached in the compound 1 and compound 16 treatment groups were significantly lower than those in the model group, but slightly higher than those in the tacrine group, indicating that compounds 1 and 16 improved the memory and cognitive functions of mice, and their effects were almost equivalent to those of tacrine.

[0140] In addition, from Figure 2 The AG results show that the trajectory of the model group mice was significantly more chaotic than that of the control group; the path of the tacrine group mice was less chaotic than that of the model group, indicating that tacrine improved the memory and cognitive function of the mice; the trajectory of the mice in the compound 1 treatment group and the compound 16 treatment group was significantly less chaotic than that of the model group and similar to that of the tacrine group, indicating that compounds 1 and 16 had a significant effect on improving the memory and cognitive function of the mice, and were comparable to the effect of tacrine.

Claims

1. A compound of Formula I or a pharmaceutically acceptable salt thereof, R is selected from substituted or unsubstituted aryl or heteroaryl groups.

2. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, R is selected from quinolinyl or isoquinolinyl, wherein The quinolinyl or isoquinolinyl group may optionally be substituted with one or more R1 groups; R1 is selected from halogen, C1-C3 alkyl, nitro, C1-C3 alkoxy, or amino.

3. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, R is selected from , , , , , , , , , , , , , , , , , , , , or .

4. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, R is selected from , , , , , , , , , , , , , or .

5. The following compounds or their pharmaceutically acceptable salts, 。 6. The following compounds or their pharmaceutically acceptable salts, 。 7. A method for preparing the compound of formula I according to claim 1, comprising the following steps: 1) In a first solvent, ammonium thiocyanate and compound i are reacted to obtain compound ii; 2) In a second solvent, the compound of formula ii and the compound of formula ii-1 are reacted to obtain the compound of formula iii; 3) In a third solvent, in the presence of basic substance A and a condensing agent, the compound of formula iii and the compound of formula iii-1 are reacted to obtain the compound of formula I; The first solvent, the second solvent, and the third solvent are each independently selected from one or more of water, toluene, xylene, dichloromethane, dichloroethane, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, and acetone; Alkaline substance A is selected from one or more of the following: triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, sodium carbonate, sodium bicarbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium hydride, and sodium amino acid. The condensing agent is selected from EDC, DIC, DCC, EDCI, DMAP, HOBt, or HOSu.

8. The preparation method according to claim 7, characterized in that, The reaction conditions in step 1) are 50~60℃ for 1~2h; the reaction conditions in step 2) are 20~25℃ for 10~12h; and the reaction conditions in step 3) are 20~25℃ for 12~18h.

9. Use of the compound according to any one of claims 1-6 in the preparation of a product for inhibiting cholinesterase activity.

10. Use of the compound according to any one of claims 1-6 in the preparation of a medicament for selectively inhibiting butyrylcholinesterase activity; preferably, use of the compound according to any one of claims 1-6 in the preparation of a medicament for the prevention or treatment of Alzheimer's disease.

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