Substituted coumarin-benzoxazole derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a substituted coumarin-benzoxazole derivative as well as a preparation method and application thereof. The derivative has a structure shown in a formula (I) or (II), R1 is selected from hydrogen, methyl, chlorine and formyloxyethyl, R2 is selected from hydrogen, methyl, trifluoromethyl and a benzene ring, and n is a positive integer ranging from 2 to 5. The preparation is realized through two-step substitution reaction, raw materials are cheap, the process is simple and convenient, and the method is suitable for industrial production. The derivative has remarkable multi-target activity, can inhibit monoamine oxidase-B and beta-amyloid protein aggregation and butyrylcholine esterase at the same time, has a good antioxidant effect, can effectively improve pathological injuries related to neurodegenerative diseases, and is low in biotoxicity and high in safety. The invention can be used for preparing drugs for treating Alzheimer's disease, Parkinson's disease and other diseases, provides a new efficient candidate molecule for the treatment of related diseases, and has important medical research value and market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a substituted coumarin-benzoxazole derivative, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a chronic central nervous system degenerative disease characterized by progressive cognitive decline, memory loss, and neurobehavioral abnormalities. Its course is irreversible and progressive, posing a significant threat to the health of the elderly population worldwide. With the increasing aging of the global population, the incidence of AD continues to rise, severely impacting patients' quality of life and imposing a heavy medical burden and economic pressure on families and society. Therefore, developing effective and safe anti-AD drugs has significant clinical value and social importance.

[0003] To date, the pathogenesis of Alzheimer's disease (AD) remains incompletely understood, involving a complex network of multifactorial and multi-target regulation. Current research confirms that AD is closely related to multiple factors, including the misfolding and aggregation of β-amyloid (Aβ) to form senile plaques, hyperphosphorylation of intracellular tau protein leading to neurofibrillary tangles (NFT), cholinergic nervous system dysfunction, oxidative stress damage, neuroinflammatory responses, mitochondrial dysfunction, imbalance of metal ion homeostasis, and abnormal activation of apoptosis pathways. These pathological factors intertwine and synergistically drive neuronal damage and death, ultimately leading to cognitive impairment.

[0004] Due to the complexity of the pathogenesis of Alzheimer's disease (AD), single-target drugs often fail to achieve ideal therapeutic effects. Traditional anti-AD drugs, such as cholinesterase inhibitors and NMDA receptor antagonists, only target a single pathological step, resulting in limited clinical efficacy and failing to slow disease progression. Therefore, the design of multi-target drugs targeting multiple key pathological targets has become a core focus and important direction in current anti-AD drug development.

[0005] Monoamine oxidase-B (MAO-B), a key enzyme involved in neurotransmitter metabolism in the brain, exhibits significantly increased activity with age and the progression of Alzheimer's disease (AD), particularly accumulating around senile plaques in the brains of AD patients. Abnormally elevated MAO-B activity leads to the depletion of neurotransmitters such as dopamine in the brain, while simultaneously generating a large number of neurotoxic reactive oxygen species (ROS), exacerbating oxidative stress damage and further promoting Aβ protein aggregation and tau protein hyperphosphorylation, forming a vicious cycle of pathological damage. Numerous studies have confirmed that MAO-B inhibitors can exert neuroprotective effects through pathways such as inhibiting enzyme activity, reducing free radical generation, and regulating neurotransmitter balance, providing an important target for AD treatment.

[0006] Oxidative stress is a key pathological step in the development of Alzheimer's disease (AD). Abnormal metabolism of substances and energy in the brain tissue of AD patients leads to an imbalance between the production and clearance of free radicals. Excessive free radicals trigger lipid peroxidation, protein oxidation, and nucleic acid damage, which in turn induce neuronal apoptosis and exacerbate the characteristic neuropathological changes of AD. Studies have shown that antioxidants can protect against AD and slow its progression through mechanisms such as scavenging reactive oxygen species, inhibiting oxidative stress responses, and reducing neuronal damage. Therefore, small molecule compounds with antioxidant activity have become an important direction for the development of anti-AD drugs.

[0007] Amyloid plaques formed by Aβ aggregation are one of the most typical pathological features of Alzheimer's disease (AD). The amyloid hypothesis posits that the imbalance between abnormal Aβ production and clearance in the brain is the core initiating factor in AD pathogenesis. Aβ originates from the abnormal metabolism of amyloid precursor protein (APP). Under normal physiological conditions, Aβ production and degradation are in dynamic equilibrium, and it plays a certain regulatory role in neuroplasticity and synapse formation. However, under pathological conditions, APP is continuously cleaved by β-secretase (BACE-1) and γ-secretase to produce excessive Aβ (mainly Aβ). 1-40 And the more neurotoxic Aβ 1-42 Simultaneously, the activity of Aβ-degrading enzymes (such as enkephalinase and insulin-degrading enzyme) decreases, leading to the accumulation of Aβ in the brain, forming insoluble oligomers and senile plaques, causing neurotoxicity, and ultimately resulting in neuronal death and cognitive impairment. Therefore, inhibiting Aβ accumulation and promoting Aβ clearance have become key targets in the development of anti-AD drugs.

[0008] Furthermore, the decline in cholinergic nervous system function is one of the important causes of cognitive impairment in AD patients. Decreased levels of acetylcholine in the brain directly affect cognitive processes such as learning and memory. Butyrylcholinesterase (BuChE), a key enzyme involved in cholinergic neurotransmitter metabolism, exhibits abnormally elevated activity, which accelerates the breakdown of acetylcholine and further exacerbates cholinergic dysfunction. Therefore, inhibiting BuChE activity and increasing acetylcholine levels in the brain is also an important therapeutic strategy for improving cognitive function in AD patients.

[0009] In summary, the pathological mechanism of AD involves multiple key targets, including abnormal MAO-B activity, Aβ aggregation, oxidative stress, and elevated cholinesterase activity. Developing multi-target compounds that can act on multiple pathological links simultaneously is expected to overcome the efficacy limitations of single-target drugs and achieve better anti-AD treatment effects. Summary of the Invention

[0010] The purpose of this invention is to provide a substituted coumarin-benzoxazole derivative, its preparation method and application. This type of compound can simultaneously inhibit MAO-B, inhibit Aβ aggregation, exert antioxidant effects and inhibit butyrylcholinesterase activity, and has low biotoxicity and high safety, providing a new candidate molecule for the development of anti-AD drugs.

[0011] The objective of this invention is achieved through the following technical solution: This invention provides a substituted coumarin-benzoxazole derivative having the structure shown in formula (I) or (II):

[0012] In formulas (I) and (II), R1 is selected from one or more of hydrogen, methyl, chloro, and formyloxyethyl; R2 is selected from hydrogen, methyl, trifluoromethyl, and benzene ring; and n is a positive integer from 2 to 5.

[0013] Furthermore, the substituted coumarin-benzoxazole derivative has any of the following structures: .

[0014] The present invention also provides a method for preparing the substituted coumarin-benzoxazole derivative, the synthetic route of which is as follows:

[0015] Specifically, the following steps are included: S1. Reacting a compound of formula (III) or (V) with Br-(CH2) n -Br undergoes a substitution reaction in an alkaline environment to give compounds of formula (IV) or (VI); S2. The compound of formula (IV) or (VI) obtained in step S1 is subjected to a substitution reaction with 4-(benzo[d]oxazol-2-yl)phenol under alkaline conditions to obtain compound (I) or compound (II). Wherein, R1 is selected from one or more of hydrogen, methyl, chloro, and formyloxyethyl; R2 is selected from hydrogen, methyl, trifluoromethyl, and benzene ring; n is a positive integer from 2 to 5.

[0016] Furthermore, the alkaline environment in step S1 is formed by one or more of cesium carbonate, potassium carbonate, and sodium carbonate, the reaction temperature is 25–60°C, and the reaction time is 2–8 h; the alkaline environment in step S2 is formed by one or more of cesium carbonate and potassium carbonate, the solvent is one or more of N,N-dimethylformamide and acetonitrile, the reaction temperature is 25–100°C, the reaction time is 2–8 h, and the reaction is purified by column chromatography.

[0017] The present invention also provides the application of a substituted coumarin-benzoxazole derivative in the preparation of a monoamine oxidase-B inhibitor.

[0018] The present invention also provides the application of a substituted coumarin-benzoxazole derivative in the preparation of butyrylcholinesterase inhibitors.

[0019] This invention also provides the application of a substituted coumarin-benzoxazole derivative in the preparation of an Aβ aggregation inhibitor.

[0020] The present invention also provides the application of a substituted coumarin-benzoxazole derivative in the preparation of antioxidants.

[0021] The present invention also provides the use of a substituted coumarin-benzoxazole derivative in the preparation of a medicament for treating Alzheimer's disease, cerebrovascular dementia, myasthenia gravis, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis.

[0022] The present invention also provides a pharmaceutical formulation comprising the aforementioned substituted coumarin-benzoxazole derivative, wherein the dosage form is selected from tablets, pills, capsules, injections, suspensions, or emulsions.

[0023] Beneficial effects: The substituted coumarin-benzoxazole derivatives provided by this invention possess a novel structural advantage. Their innovative structure endows them with a multi-target synergistic mechanism, simultaneously targeting four key pathological targets: monoamine oxidase-B (MAO-B) activity, β-amyloid (Aβ) aggregation, butyrylcholinesterase activity, and oxidative stress. This comprehensively covers the core pathogenesis of neurodegenerative diseases such as Alzheimer's disease. Experimental data confirm that these derivatives have significant inhibitory activity against MAO-B, with representative compounds achieving an inhibition rate of up to 73.6%, superior to the positive control drug; and against Aβ... 1-42 The self-aggregation inhibition rate reached up to 96.2%, effectively blocking the formation of amyloid plaques. Its in vitro antioxidant activity was comparable to curcumin, efficiently scavenging reactive oxygen species and reducing oxidative stress damage. Simultaneously, it exhibited the highest inhibition rate against butyrylcholinesterase (67.8%), improving the functional decline of the cholinergic nervous system. This multi-target synergistic effect enabled these compounds to effectively reverse scopolamine-induced memory impairment in mice, significantly enhancing the learning and memory abilities of model animals. This provides a more comprehensive and efficient solution for the treatment of neurodegenerative diseases, overcoming the limitations of traditional single-target drugs. Furthermore, these derivatives exhibit extremely low biotoxicity, with a half-maximal inhibitory concentration (IC50) for nerve cells (SH-SY5Y). 50 The concentration reached up to 117.6 μM, which is far higher than the effective concentration, demonstrating significant safety advantages and laying a solid foundation for its clinical application.

[0024] The preparation method of this invention has significant industrial application value. Its synthetic route is scientifically and rationally designed, using readily available and inexpensive coumarin intermediates and benzoxazole compounds as raw materials. The target product can be efficiently prepared through a two-step substitution reaction. The reaction conditions are mild (25–100 °C) and the reaction time is short (2–8 h), requiring no complex reaction equipment. Steps S1 and S2 both use conventional alkaline reagents to construct the reaction environment, and commonly used industrial solvents such as N,N-dimethylformamide and acetonitrile are selected as solvents. Post-processing only requires column chromatography to obtain high-purity products. The overall process is simple to operate, cost-controllable, and has minimal environmental pollution, making it suitable for large-scale industrial production. Furthermore, pharmaceutical formulations developed based on these derivatives can be made into various dosage forms such as tablets, pills, and capsules to meet the needs of different clinical scenarios. Combined with its significant therapeutic effects and high safety, it not only has important medical research value but also fills the clinical gap in the treatment of neurodegenerative diseases, possessing broad market application prospects. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 In Experimental Example 6 of this invention, the MTT assay was used to test the effects of compounds Gn-4d and Gn-5d on Aβ. 1-42 Statistical graph of protective effect against induced SH-SY5Y cell damage; Figure 2 The figure shows the results of the passive avoidance experiment of the AD model mice in Experiment Example 7 of this invention. (A) Delay time (s); (B) Error count statistics; data are expressed as mean ± standard deviation (n=8). Compared with the control group, # p<0.01, *p<0.01, **p<0.05. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Example 1 Synthesis of Gn-2a, a substituted coumarin-benzoxazole derivative

[0033] In a 25 mL round-bottom flask, add 6 mL of DMF, 0.13 g (0.74 mmol) of 4-(benzo[d]oxazol-2-yl)phenol, and 0.20 g (1.24 mmol) of anhydrous potassium carbonate. After stirring the mixture at room temperature for 30 min, add the coumarin intermediate 7-(2-bromoethoxy)-2 H -Crotene-2-one (0.2 g, 0.74 mmol) was stirred at room temperature, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was evaporated to dryness under reduced pressure, water was added and stirred at room temperature for 2 h to allow the solid to fully precipitate. The solid was then filtered, dried, and separated by silica gel column chromatography to obtain the coumarin-benzoxazole derivative Gn-2a, with a yield of 32.7%.

[0034] 1H NMR (500 MHz, DMSO) δ 8.18 – 8.15 (m, 2H), 8.02 (d, J = 9.5 Hz,1H), 7.81 – 7.75 (m, 2H), 7.67 (d, J = 8.6 Hz, 1H), 7.42 – 7.38 (m, 2H), 7.26– 7.22 (m, 2H), 7.10 (d, J = 2.4 Hz, 1H), 7.03 (dd, J = 8.6, 2.5 Hz, 1H), 6.32 (d, J = 9.5 Hz, 1H), 4.50 (s, 4H). 13 C NMR (151 MHz, CDCl3) δ 163.09,162.00, 161.49, 161.19, 155.88, 150.68, 143.39, 142.25, 129.43(2C), 128.82,124.67, 124.46, 119.89, 119.64, 114.86(2C), 113.20, 112.81, 112.67, 110.41,101.52, 64.88, 64.32. ESI-MS m / z: 400.12 [M+H] + .

[0035] Example 2 Synthesis of Gn-3a, a substituted coumarin-benzoxazole derivative

[0036] The difference from Example 1 is that the raw material used in this example is 7-(3-bromopropoxy)-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, yielding compound 3a, yield: 72.0%.

[0037] 1 H NMR (600 MHz, CDCl3) δ 8.18 (dd, J = 7.1, 4.9 Hz, 2H), 7.72 (dt, J = 6.8, 3.2 Hz, 1H), 7.61 (d, J = 9.5 Hz, 1H), 7.54 (dd, J= 6.3, 2.8 Hz, 1H),7.38 – 7.29 (m, 3H), 7.06 – 7.01 (m, 2H), 6.87 – 6.82 (m, 2H), 6.24 (d, J =9.5 Hz, 1H), 4.24 (dt, J = 8.3, 5.9 Hz, 4H), 2.33 (p, J = 6.0 Hz, 2H). 13 C NMR(151 MHz, CDCl3) δ 163.09, 162.00, 161.49, 161.19, 155.88, 150.68, 143.39,142.25, 129.43(2C), 128.82, 124.67, 124.46, 119.89, 119.64, 114.86(2C),113.20, 112.81, 112.67, 110.41, 101.52, 64.88, 64.32, 28.98. ESI-MS m / z:414.13 [M+H] + .

[0038] Example 3 Synthesis of Gn-4a, a substituted coumarin-benzoxazole derivative

[0039] The difference from Example 1 is that the raw material used in this example is 7-(4-bromobutoxy)-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, yielding compound 4a, yield: 45.6%.

[0040] 1 H NMR (600 MHz, CDCl3) δ 8.20 – 8.17 (m, 2H), 7.74 – 7.72 (m, 1H),7.62 (d, J = 9.5 Hz, 1H), 7.56 – 7.53 (m, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.32(ddd, J = 6.5, 3.4, 1.8 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 6.84 – 6.81 (m,2H), 6.24 (d,J = 9.4 Hz, 1H), 4.11 (dd, J = 12.0, 5.7 Hz, 4H), 2.04 (h, J =3.5 Hz, 4H). 13 C NMR (151 MHz, CDCl3) δ 163.16, 162.19, 161.67, 161.25,155.92, 150.68, 143.42, 142.28, 129.42(2C), 128.78, 124.64, 124.45, 119.72,119.63, 114.83(2C), 113.09, 112.92, 112.55, 110.40, 101.37, 68.09, 67.58,25.87, 25.80. ESI-MS m / z: 428.15 [M+H] + .

[0041] Example 4 Synthesis of Gn-5a, a substituted coumarin-benzoxazole derivative

[0042] The difference from Example 1 is that the raw material used in this example is 7-(5-bromopentoxy)-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crone-2-one, other parameters and operations are as described in Example 1, to obtain compound Gn-4a, yield: 21.0%.

[0043] 1 H NMR (600 MHz, CDCl3) δ 8.20 – 8.14 (m, 2H), 7.73 (dd, J = 6.5, 2.8Hz, 1H), 7.61 (d, J = 9.5 Hz, 1H), 7.57 – 7.52 (m, 1H), 7.36 – 7.28 (m, 3H),7.01 (d, J = 8.5 Hz, 2H), 6.84 – 6.78 (m, 2H), 6.23 (d, J = 9.5 Hz, 1H), 4.06(dt, J = 18.1, 6.3 Hz, 4H), 1.91 (p, J= 6.9 Hz, 4H), 1.72 – 1.66 (m, 2H). 13 CNMR (151 MHz, CDCl3) δ 163.20, 162.29, 161.79, 161.26, 155.92, 150.68,143.44, 142.30, 129.40(2C), 128.76, 124.62, 124.44, 119.62, 119.61, 114.84(2C), 113.01, 112.95, 112.48, 110.40, 101.35, 68.36, 67.89, 29.72, 28.88,28.75, 22.71. ESI-MS m / z: 442.16 [M+H] + .

[0044] Example 5 Synthesis of Gn-2b, a substituted coumarin-benzoxazole derivative

[0045] The difference from Example 1 is that the raw material used in this example is 7-(2-bromoethoxy)-4-methyl-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain compound 2b, yield: 41.2%.

[0046] 1 H NMR (600 MHz, DMSO- d 6 )) δ 8.16 (d, J = 8.6 Hz, 2H), 7.80 – 7.74 (m,2H), 7.72 (d, J = 8.7 Hz, 1H), 7.41 – 7.38 (m, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 2.4 Hz, 1H), 7.04 (dd, J = 8.8, 2.5 Hz, 1H), 6.24 (s, 1H), 4.49(d, J = 3.1 Hz, 4H). 13C NMR (151 MHz, CDCl3) δ 163.09, 161.82, 161.50,161.26, 155.27, 152.50, 150.68, 142.27, 129.43(2C), 125.58, 124.66, 124.46,119.89, 119.64, 114.86(2C), 113.72, 112.48, 112.06, 110.41, 101.53, 64.83,64.35, 18.67. ESI-MS m / z: 414.13 [M+H] + .

[0047] Example 6 Synthesis of Gn-3b, a substituted coumarin-benzoxazole derivative

[0048] The difference from Example 1 is that the raw material used in this example is 7-(3-bromopropoxy)-4-methyl-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, yielding compound 3b, yield: 33.3%.

[0049] 1 H NMR (600 MHz, CDCl3) δ 8.21 (d, J = 8.4 Hz, 2H), 7.78 – 7.72 (m,1H), 7.60 – 7.54 (m, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.34 (hept, J = 5.5 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.91 – 6.84 (m, 2H), 6.14 (s, 1H), 4.27 (dt, J = 11.5, 6.0 Hz, 4H), 2.38 (d, J = 22.5 Hz, 5H). 13C NMR (151 MHz, CDCl3) δ163.09, 161.82, 161.50, 161.26, 155.27, 152.50, 150.68, 142.27, 129.43(2C),125.58, 124.66, 124.46, 119.89, 119.64, 114.86(2C), 113.72, 112.48, 112.06,110.41, 101.53, 64.83, 64.35, 29.00, 18.67. ESI-MS m / z: 428.15 [M+H] + .

[0050] Example 7 Synthesis of Gn-4b, a substituted coumarin-benzoxazole derivative

[0051] The difference from Example 1 is that the raw material used in this example is 7-(4-bromobutoxy)-4-methyl-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, yielding compound 4b with a yield of 35.3%.

[0052] 1 H NMR (600 MHz, CDCl3) δ 8.18 (d, J = 8.3 Hz, 2H), 7.75 – 7.70 (m,1H), 7.57 – 7.52 (m, 1H), 7.47 (d, J = 8.7 Hz, 1H), 7.32 (dq, J = 6.5, 3.8Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.86 – 6.80 (m, 2H), 6.12 (s, 1H), 4.14 –4.08 (m, 4H), 2.38 (s, 3H), 2.06 – 2.01 (m, 4H). 13C NMR (151 MHz, CDCl3) δ163.16, 161.99, 161.68, 161.34, 155.29, 152.57, 150.67, 142.27, 129.41(2C),125.55, 124.63, 124.45, 119.69, 119.62, 114.83(2C), 113.58, 112.60, 111.94,110.40, 101.37, 68.04, 67.60, 25.87, 25.80, 18.67. ESI-MS m / z: 442.17 [M+H] + .

[0053] Example 8 Synthesis of Gn-5b, a substituted coumarin-benzoxazole derivative

[0054] The difference from Example 1 is that the raw material used in this example is 7-(5-bromopentoxy)-4-methyl-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, yielding compound 5b, yield: 38.8%.

[0055] 1 H NMR (600 MHz, CDCl3) δ 8.17 (d, J = 8.6 Hz, 2H), 7.74 – 7.71 (m,1H), 7.55 – 7.52 (m, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.31 (tt, J = 7.3, 5.5Hz, 2H), 7.01 (d, J = 8.6 Hz, 2H), 6.84 (dd, J = 8.8, 2.5 Hz, 1H), 6.79 (d, J = 2.6 Hz, 1H), 6.11 (s, 1H), 4.06 (dt, J = 18.6, 6.2 Hz, 4H), 2.37 (s, 3H), 1.90 (q, J = 7.0 Hz, 4H), 1.72 – 1.67 (m, 2H). 13C NMR (151 MHz, CDCl3) δ163.20, 162.09, 161.79, 161.36, 155.29, 152.59, 150.67, 142.28, 129.39(2C),125.51, 124.61, 124.43, 119.60, 119.58, 114.83(2C), 113.51, 112.63, 111.88,110.39, 101.35, 68.30, 67.89, 28.88, 28.75, 22.70, 18.66. ESI-MS m / z: 456.18[M+H] + .

[0056] Example 9 Synthesis of Gn-3c, a substituted coumarin-benzoxazole derivative

[0057] The difference from Example 1 is that the raw material used in this example is 4-(3-bromopropoxy)-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, yielding compound 3c, yield: 27.0%.

[0058] 1 H NMR (600 MHz, CDCl3) δ 8.19 (dt, J = 8.7, 2.9 Hz, 2H), 7.81 (dt, J = 6.2, 2.8 Hz, 1H), 7.73 (dt, J = 6.0, 2.6 Hz, 1H), 7.54 (dq, J = 9.5, 4.4Hz, 2H), 7.32 (dd, J = 6.7, 3.5 Hz, 3H), 7.04 (dt, J = 8.7, 2.9 Hz, 2H), 5.73(t, J = 2.9 Hz, 1H), 4.36 (dd, J = 7.1, 4.1 Hz, 2H), 4.28 (dd, J = 6.9, 4.0Hz, 2H), 2.44 (p, J = 5.6 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 165.42, 163.01,162.80, 161.26, 153.38, 150.69, 142.25, 132.49, 129.47(2C), 124.71, 124.49,123.92, 122.89, 120.13, 119.68, 116.88, 115.63, 114.83(2C), 110.42, 90.79,65.81, 64.10, 29.72. ESI-MS m / z: 414.13 [M+H] + .

[0059] Example 10 Synthesis of Gn-4c, a substituted coumarin-benzoxazole derivative

[0060] The difference from Example 1 is that the raw material used in this example is 4-(2-bromobutoxy)-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, yielding compound 4c, yield: 69.2%.

[0061] 1 H NMR (600 MHz, CDCl3) δ 8.21 (dd, J = 8.3, 4.4 Hz, 2H), 7.78 (dt, J = 28.0, 5.9 Hz, 2H), 7.60 – 7.51 (m, 2H), 7.38 – 7.30 (m, 3H), 7.04 (dd, J =8.7, 4.3 Hz, 2H), 5.73 – 5.69 (m, 1H), 4.22 (dt, J = 43.7, 5.7 Hz, 4H), 2.21– 2.08 (m, 4H). 13C NMR (151 MHz, CDCl3) δ 165.56, 163.09, 162.91, 161.52,153.36, 150.69, 142.28, 132.42, 129.44(2C), 124.67, 124.47, 123.88, 122.93,119.85, 119.65, 116.82, 115.69, 114.81(2C), 110.41, 90.56, 68.97, 67.37,25.89, 25.44.ESI-MS m / z: 428.15 [M+H] + .

[0062] Example 11 Synthesis of Gn-5c, a substituted coumarin-benzoxazole derivative

[0063] The difference from Example 1 is that the raw material used in this example is 4-(5-bromopentoxy)-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crone-2-one, other parameters and operations are as described in Example 1, yielding compound 5c, yield: 42%.

[0064] 1 H NMR (600 MHz, CDCl3) δ 8.23 ​​– 8.14 (m, 2H), 7.84 – 7.79 (m, 1H),7.73 (t, J = 5.4 Hz, 1H), 7.55 (dd, J = 7.6, 4.2 Hz, 2H), 7.36 – 7.29 (m,3H), 7.05 – 6.99 (m, 2H), 5.68 (d, J = 4.2 Hz, 1H), 4.17 (q, J = 5.9 Hz, 2H), 4.10 (q, J = 5.8 Hz, 2H), 2.01 (q, J = 6.7 Hz, 2H), 1.94 (q, J = 6.7 Hz, 2H), 1.76 (dq, J = 14.6, 6.9 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 165.64, 163.15,162.96, 161.72, 153.38, 150.68, 142.29, 132.40, 129.41(2C), 124.64, 124.45,123.88, 122.98, 119.70, 119.63, 116.82, 115.77, 114.82(2C), 110.39, 90.50,69.12, 67.73, 28.81, 28.28, 22.72. ESI-MS m / z: 442.17 [M+H] + .

[0065] Example 12 Synthesis of Gn-2d, a substituted coumarin-benzoxazole derivative

[0066] The difference from Example 1 is that the raw material used in this example is 7-(2-bromoethoxy)-3,4-dimethyl-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain compound 2d, yield: 58.6%.

[0067] 1 H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 8.7 Hz, 2H), 7.80 – 7.74 (m,1H), 7.61 – 7.53 (m, 2H), 7.38 – 7.34 (m, 2H), 7.12 (dd, J = 9.3, 2.7 Hz,2H), 6.97 – 6.88 (m, 2H), 4.52 – 4.41 (m, 4H), 2.41 (s, 3H), 2.22 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 163.10, 162.40, 161.53, 160.69, 153.54, 150.68,146.18, 142.28, 129.41(2C), 125.30, 124.64, 124.45, 119.86, 119.64, 119.05,114.87(2C), 114.33, 112.26, 110.40, 101.24, 64.72, 64.41, 15.07, 13.17. ESI-MS m / z: 428.15 [M+H] + .

[0068] Example 13 Synthesis of Gn-3d, a substituted coumarin-benzoxazole derivative

[0069] The difference from Example 1 is that the raw material used in this example is 7-(3-bromopropoxy)-3,4-dimethyl-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain compound 3d, yield: 42.8%.

[0070] 1 H NMR (600 MHz, CDCl3) δ 8.21 – 8.15 (m, 2H), 7.72 (td, J = 5.2, 2.7Hz, 1H), 7.54 (dt, J = 6.6, 2.8 Hz, 1H), 7.49 – 7.45 (m, 1H), 7.35 – 7.29 (m,2H), 7.06 – 7.01 (m, 2H), 6.87 – 6.79 (m, 2H), 4.24 (dt, J = 19.1, 5.6 Hz,4H), 2.38 – 2.29 (m, 5H), 2.20 – 2.15 (m, 3H). 13C NMR (151 MHz, CDCl3) δ163.10, 162.40, 161.53, 160.69, 153.54, 150.68, 146.18, 142.28, 129.41(2C),125.30, 124.64, 124.45, 119.86, 119.64, 119.05, 114.87(2C), 114.33, 112.26,110.40, 101.24, 64.72, 64.41, 29.04, 15.07, 13.17. ESI-MS m / z: 442.17 [M+H] + .

[0071] Example 14 Synthesis of Gn-4d, a substituted coumarin-benzoxazole derivative

[0072] The difference from Example 1 is that the raw material used in this example is 7-(4-bromobutoxy)-3,4-dimethyl-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, compound 4d was obtained, yield: 45.3%.

[0073] 1 H NMR (600 MHz, CDCl3) δ 8.21 – 8.15 (m, 2H), 7.73 (dd, J = 7.2, 3.8Hz, 1H), 7.51 (ddd, J = 45.4, 8.6, 4.0 Hz, 2H), 7.35 – 7.29 (m, 2H), 7.05 –6.99 (m, 2H), 6.85 – 6.78 (m, 2H), 4.11 (dd, J = 20.6, 4.8 Hz, 4H), 2.35 (d, J = 3.7 Hz, 3H), 2.17 (d, J = 3.8 Hz, 3H), 2.03 (d, J = 5.3 Hz, 4H). 13C NMR(151 MHz, CDCl3) δ 163.17, 162.45, 161.69, 160.87, 153.58, 150.6 9, 146.24,142.30, 129.41(2C), 125.27, 124.62, 124.44, 119.69, 119.63, 118.95, 114.83(2C), 114.20, 112.39, 110.39, 101.09, 67.93, 67.62, 25.90, 25.83, 15.07,13.16. ESI-MS m / z: 456.18 [M+H] + .

[0074] Example 15 Synthesis of Gn-5d, a substituted coumarin-benzoxazole derivative

[0075] The difference from Example 1 is that the raw material used in this example is 7-(5-bromopentoxy)-3,4-dimethyl-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, compound 4d was obtained, yield: 55.3%.

[0076] 1 H NMR (400 MHz, CDCl3) δ 8.19 – 8.15 (m, 2H), 7.75 – 7.70 (m, 1H),7.56 – 7.52 (m, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.34 – 7.28 (m, 2H), 7.03 –6.98 (m, 2H), 6.84 – 6.76 (m, 2H), 4.05 (dt, J = 17.0, 6.3 Hz, 4H), 2.34 (d, J = 1.0 Hz, 3H), 2.16 (s, 3H), 1.94 – 1.86 (m, 4H), 1.69 (dddd, J = 14.9, 9.4, 6.4, 3.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 163.20, 162.48, 161.79,160.95, 153.55, 150.66, 146.29, 142.29, 129.38(2C), 125.24, 124.60, 124.43,119.61, 119.55, 118.85, 114.82(2C), 114.10, 112.41, 110.39, 101.04, 68.18,67.90, 28.89, 28.79, 22.72, 15.08, 13.16. ESI-MS m / z: 470.20 [M+H] + .

[0077] Example 16 Synthesis of Gn-2e, a substituted coumarin-benzoxazole derivative

[0078] The difference from Example 1 is that the raw material used in this example is 7-(2-bromoethoxy)-4-phenyl-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crone-2-one, other parameters and operations are as described in Example 1, yielding compound 2e in 31.3%.

[0079] 1 H NMR (600 MHz, CDCl3) δ 8.23 ​​– 8.18 (m, 2H), 7.76 – 7.71 (m, 1H),7.55 (dt, J = 7.5, 2.3 Hz, 1H), 7.51 (dq, J = 4.6, 2.5 Hz, 3H), 7.46 – 7.42(m, 2H), 7.42 – 7.38 (m, 1H), 7.33 – 7.25 (m, 2H), 7.07 (dt, J = 8.9, 2.2 Hz, 2H), 6.95 (q, J = 2.3 Hz, 1H), 6.85 (dt, J = 9.3, 2.6 Hz, 1H), 6.25 – 6.22(m, 1H), 4.45 – 4.42 (m, 4H). 13C NMR (151 MHz, CDCl3) δ 162.98, 161.66,161.13, 155.94, 155.75, 150.70, 142.26, 135.50, 129.66, 129.46(2C), 128.88(2C), 128.40(2C), 128.14, 124.74, 124.50, 120.35, 119.70, 115.00(2C), 112.95,112.74, 112.19, 110.44, 101.85, 66.94, 66.35. ESI-MS m / z: 476.15 [M+H] + .

[0080] Example 17 Synthesis of Gn-3e, a substituted coumarin-benzoxazole derivative

[0081] The difference from Example 1 is that the raw material used in this example is 7-(3-bromopropoxy)-4-phenyl-2- H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, yielding compound 3e, yield: 73.0%.

[0082] 1 H NMR (400 MHz, CDCl3) δ 8.21 – 8.16 (m, 2H), 7.75 – 7.71 (m, 1H), 7.56 – 7.53 (m, 1H), 7.53 – 7.48 (m, 3H), 7.45 – 7.40 (m, 2H), 7.38 (d, J =8.9 Hz, 1H), 7.35 – 7.28 (m, 2H), 7.06 – 7.02 (m, 2H), 6.92 (d, J = 2.5 Hz, 1H), 6.80 (dd, J = 8.9, 2.5 Hz, 1H), 6.21 (s, 1H), 4.26 (t, J = 6.0 Hz, 4H), 2.35 (p, J = 5.9 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 163.09, 161.97, 161.49,161.23, 155.99, 155.79, 150.68, 142.26, 135.55, 129.62, 129.43(2C), 128.85(2C), 128.40(2C), 128.06, 124.67, 124.47, 119.65, 114.86(2C), 112.55, 111.97,110.42, 101.75, 64.88, 64.31, 28.98. ESI-MS m / z: 490.17 [M+H] + .

[0083] Example 18 Synthesis of Gn-4e, a substituted coumarin-benzoxazole derivative

[0084] The difference from Example 1 is that the raw material used in this example is 7-(4-bromobutoxy)-4-phenyl-2- H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, yielding compound 4e, yield: 66.5%.

[0085] 1 H NMR (600 MHz, CDCl3) δ 8.18 (ddd, J = 9.0, 4.1, 1.8 Hz, 2H), 7.73(dt, J = 5.3, 3.0 Hz, 1H), 7.57 – 7.48 (m, 4H), 7.43 (dd, J = 6.0, 3.1 Hz, 2H), 7.35 (ddt, J = 29.6, 7.2, 2.4 Hz, 3H), 7.02 (ddd, J = 8.9, 4.1, 1.8 Hz,2H), 6.89 (p, J = 2.2 Hz, 1H), 6.78 (ddt, J = 8.5, 4.4, 2.2 Hz, 1H), 6.25 –6.17 (m, 1H), 4.13 (t, J = 4.7 Hz, 4H), 2.04 (t, J = 4.1 Hz, 4H).13 C NMR (151MHz, CDCl3) δ 163.16, 162.15, 161.67, 161.25, 156.03, 155.83, 150.69, 142.30,135.60, 129.60, 129.42(2C), 128.84(2C), 128.40(2C), 128.01, 124.63, 124.44,119.64, 114.83(2C), 112.65, 111.87, 110.40, 101.61, 68.11, 67.59, 25.84(2C).ESI-MS m / z: 504.18 [M+H] + .

[0086] Example 19 Synthesis of Gn-5e, a substituted coumarin-benzoxazole derivative

[0087] The difference from Example 1 is that the raw material used in this example is 7-(5-bromopentoxy)-4-phenyl-2- H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, compound 4d was obtained, yield: 26.9%.

[0088] 1 H NMR (400 MHz, CDCl3) δ 8.21 – 8.15 (m, 2H), 7.76 – 7.71 (m, 1H), 7.57 – 7.53 (m, 1H), 7.52 – 7.48 (m, 3H), 7.43 (ddd, J = 6.1, 2.7, 1.5 Hz,2H), 7.37 (d, J = 8.9 Hz, 1H), 7.34 – 7.28 (m, 2H), 7.04 – 6.99 (m, 2H), 6.88(d, J = 2.5 Hz, 1H), 6.78 (dd, J = 8.9, 2.5 Hz, 1H), 6.21 (s, 1H), 4.07 (q, J = 6.1 Hz, 4H), 1.91 (dtt, J = 8.7, 6.3, 3.2 Hz, 4H), 1.70 (ddt, J= 9.0, 6.7, 2.9 Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ 163.20, 162.25, 161.79, 161.29,156.03, 155.85, 150.68, 142.28, 135.61, 129.59, 129.41(2C), 128.84(2C),128.40(2C), 127.98, 124.62, 124.44, 119.62, 119.59, 114.84(2C), 112.69,112.45, 111.81, 110.40, 101.59, 68.38, 67.89, 28.90, 28.76, 22.72. ESI-MS m / z: 518.20 [M+H] + .

[0089] Example 20 Synthesis of Gn-2f, a substituted coumarin-benzoxazole derivative

[0090] The difference from Example 1 is that the coumarin intermediate in this example is 7-(2-bromoethoxy)-3-chloro-4-methyl-2- H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain the substituted coumarin-benzoxazole derivative Gn-2f, yield: 35.0%.

[0091] 1 H NMR (600 MHz, DMSO- d 6 )) δ 8.16 (d, J = 8.4 Hz, 2H), 7.81 (d, J =8.9 Hz, 1H), 7.79 – 7.75 (m, 2H), 7.41 – 7.38 (m, 2H), 7.23 (d, J = 8.5 Hz, 2H), 7.16 (d, J = 2.5 Hz, 1H), 7.10 (d, J = 8.8 Hz, 1H), 4.50 (dd, J = 14.2,5.1 Hz, 4H), 2.56 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 163.06, 161.72, 161.47,157.39, 153.09, 150.68, 147.91, 142.26, 129.42(2C), 125.92, 124.67, 124.46,119.92, 119.65, 117.89, 114.85(2C), 113.41, 113.15, 110.41, 101.43, 64.95,64.29, 16.17. ESI-MS m / z: 448.10 [M+H] + .

[0092] Example 21 Synthesis of Gn-3f, a substituted coumarin-benzoxazole derivative

[0093] The difference from Example 1 is that the coumarin intermediate in this example is 7-(3-bromopropoxy)-3-chloro-4-methyl-2- H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain the substituted coumarin-benzoxazole derivative Gn-3f, yield: 48.3%.

[0094] 1 H NMR (600 MHz, CDCl3) δ 8.20 – 8.15 (m, 2H), 7.72 (dq, J = 5.0, 2.6Hz, 1H), 7.56 – 7.47 (m, 2H), 7.36 – 7.28 (m, 2H), 7.03 (ddt, J = 6.5, 4.4,2.2 Hz, 2H), 6.92 – 6.87 (m, 1H), 6.86 – 6.82 (m, 1H), 4.25 (dt, J = 11.2,5.5 Hz, 4H), 2.54 – 2.49 (m, 3H), 2.34 (h, J = 5.7 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 163.06, 161.72, 161.47, 157.39, 153.09, 150.68, 147.91, 142.26,129.42(2C), 125.92, 124.67, 124.46, 119.92, 119.65, 117.89, 114.85(2C),113.41, 113.15, 110.41, 101.43, 64.95, 64.29, 28.98, 16.17. ESI-MS m / z:462.11 [M+H] + .

[0095] Example 22 Synthesis of Gn-4f, a substituted coumarin-benzoxazole derivative

[0096] The difference from Example 1 is that the coumarin intermediate in this example is 7-(4-bromobutoxy)-3-chloro-4-methyl-2- H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain the substituted coumarin-benzoxazole derivative Gn-4f, yield: 75.3%.

[0097] 1 H NMR (400 MHz, CDCl3) δ 8.22 – 8.16 (m, 2H), 7.76 – 7.71 (m, 1H),7.58 – 7.54 (m, 1H), 7.51 (d, J = 8.9 Hz, 1H), 7.36 – 7.29 (m, 2H), 7.05 –7.00 (m, 2H), 6.89 (dd, J = 8.9, 2.5 Hz, 1H), 6.83 (d, J = 2.5 Hz, 1H), 4.13(dt, J = 7.4, 5.3 Hz, 4H), 2.54 (s, 3H), 2.11 – 2.00 (m, 4H). 13C NMR (101MHz, CDCl3) δ 163.15, 161.90, 161.64, 157.49, 153.15, 150.68, 147.99, 142.28,129.42(2C), 125.90, 124.65, 124.46, 119.73, 119.64, 117.80, 114.82(2C),113.31, 110.41, 101.26, 68.16, 67.56, 29.73, 25.85, 16.20. ESI-MS m / z: 476.13 [M+H] + .

[0098] Example 23 Synthesis of Gn-5f, a substituted coumarin-benzoxazole derivative

[0099] The difference from Example 1 is that the coumarin intermediate in this example is 7-(5-bromopentoxy)-3-chloro-4-methyl-2- H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain the substituted coumarin-benzoxazole derivative Gn-5f, yield: 76.1%.

[0100] 1 H NMR (600 MHz, CDCl3) δ 8.17 (dt, J = 9.1, 2.2 Hz, 2H), 7.74 – 7.70(m, 1H), 7.56 – 7.52 (m, 1H), 7.48 (dt, J = 9.3, 2.3 Hz, 1H), 7.35 – 7.29 (m,2H), 7.03 – 6.99 (m, 2H), 6.89 – 6.85 (m, 1H), 6.79 (q, J = 2.6 Hz, 1H), 4.09– 4.03 (m, 4H), 2.54 – 2.49 (m, 3H), 1.90 (q, J = 7.5 Hz, 4H), 1.72 – 1.68(m, 2H). 13C NMR (151 MHz, CDCl3) δ 163.17, 162.00, 161.77, 157.46, 153.13,150.67, 147.98, 142.29, 129.39(2C), 125.85, 124.62, 124.44, 119.61, 117.70,114.83(2C), 113.29, 113.19, 110.39, 101.24, 68.42, 67.87, 28.87, 28.73,22.70, 16.16. ESI-MS m / z: 490.14 [M+H] + .

[0101] Example 24 Synthesis of Gn-2g, a substituted coumarin-benzoxazole derivative

[0102] The difference from Example 1 is that the coumarin intermediate in this example is 7-(2-bromoethoxy)-2-oxo-2- H -Ethyl chromene-3-carboxylate instead of 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain the substituted coumarin-benzoxazole derivative Gn-2g, yield: 35.0%.

[0103] 1 H NMR (500 MHz, DMSO- d 6 )) δ 8.75 (s, 1H), 8.18 – 8.15 (m, 2H), 7.88(d, J = 8.7 Hz, 1H), 7.77 (dq, J = 5.1, 2.2 Hz, 2H), 7.41 – 7.39 (m, 2H), 7.25 – 7.22 (m, 2H), 7.15 (d, J = 2.4 Hz, 1H), 7.09 (dd, J = 8.7, 2.4 Hz, 1H), 4.55 (dd, J = 6.0, 2.7 Hz, 2H), 4.50 (dd, J = 5.7, 3.0 Hz, 2H), 4.28 (t, J = 7.1 Hz, 2H), 1.31 (t, J= 7.1 Hz, 3H). 13 C NMR (151 MHz, DMSO- d 6 )) δ164.19, 163.30, 162.78, 161.61, 157.39, 156.75, 150.61, 149.63, 142.14,132.21, 129.67(2C), 125.53, 125.22, 119.94, 119.58, 115.84(2C), 114.14,114.01, 112.15, 111.22, 101.40, 67.77, 66.88, 61.43, 14.60. ESI-MS m / z: 472.14 [M+H] + .

[0104] Example 25 Synthesis of the substituted coumarin-benzoxazole derivative Gn-3g

[0105] The difference from Example 1 is that the coumarin intermediate in this example is 7-(3-bromopropoxy)-2-oxo-2- H -Ethyl chromene-3-carboxylate instead of 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain the substituted coumarin-benzoxazole derivative Gn-3g, yield: 54.5%.

[0106] 1 H NMR (400 MHz, CDCl3) δ 8.49 (s, 1H), 8.22 – 8.16 (m, 2H), 7.76 –7.70 (m, 1H), 7.57 – 7.52 (m, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.35 – 7.29 (m,2H), 7.06 – 7.01 (m, 2H), 6.89 (dd, J = 8.7, 2.4 Hz, 1H), 6.83 (d, J = 2.3Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 4.26 (q, J = 6.1 Hz, 4H), 2.35 (p, J = 6.0Hz, 2H), 1.39 (t,J = 7.1 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 164.28, 163.44,163.05, 161.41, 157.51, 157.12, 148.93, 142.24, 130.77, 129.44(2C), 124.68,124.47, 119.96, 119.65, 114.84(2C), 114.23, 113.81, 111.75, 110.42, 101.00,77.25, 65.17, 64.16, 61.73, 28.89, 14.30. ESI-MS m / z: 486.16 [M+H] + .

[0107] Example 26 Synthesis of Gn-5g, a substituted coumarin-benzoxazole derivative

[0108] The difference from Example 1 is that the coumarin intermediate in this example is 7-(5-bromopentoxy)-2-oxo-2- H -Ethyl chromene-3-carboxylate instead of 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain the substituted coumarin-benzoxazole derivative Gn-5g, yield: 47.9%.

[0109] 1 H NMR (400 MHz, CDCl3) δ 8.48 (s, 1H), 8.20 – 8.15 (m, 2H), 7.75 –7.70 (m, 1H), 7.56 – 7.52 (m, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.34 – 7.29 (m,2H), 7.03 – 6.98 (m, 2H), 6.87 (dd, J = 8.7, 2.3 Hz, 1H), 6.79 (d, J = 2.3Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 4.07 (td, J = 6.3, 2.6 Hz, 4H), 1.91 (q, J = 6.6 Hz, 4H), 1.74 (d,J = 7.6 Hz, 1H), 1.68 (d, J = 8.3 Hz, 1H), 1.39 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 164.60, 163.47, 163.17, 161.75,157.57, 157.23, 150.66, 149.02, 142.26, 130.72, 129.40(2C), 124.63, 124.45,119.61, 114.82(2C), 113.98, 113.96, 111.55, 110.40, 100.78, 77.27, 68.66,67.83, 61.71, 28.85, 28.65, 22.69, 14.31. ESI-MS m / z: 514.19 [M+H] + Example 27 Synthesis of Gn-2i, a substituted coumarin-benzoxazole derivative

[0110] The difference from Example 1 is that the coumarin intermediate in this example is 7-(2-bromoethoxy)-4-(trifluoromethyl)-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, with other parameters and procedures as described in Example 1, yielded the substituted coumarin-benzoxazole derivative Gn-2i, in 20.3% yield. 1 H NMR (500 MHz, DMSO- d 6 )) δ 8.19 – 8.14 (m, 2H), 7.79 – 7.75 (m, 2H),7.65 (dt, J = 9.1, 2.0 Hz, 1H), 7.42 – 7.37 (m, 2H), 7.26 (d, J = 2.6 Hz, 1H), 7.24 (d, J = 2.0 Hz, 1H), 7.23 (d, J = 2.2 Hz, 1H), 7.14 (dd, J = 9.0,2.5 Hz, 1H), 6.88 (s, 1H), 4.54 (dd,J = 6.1, 2.6 Hz, 2H), 4.50 (dd, J = 6.0, 2.8 Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ 163.06, 162.71, 161.42, 159.40,156.32, 150.68, 142.23, 141.73, 129.45(2C), 126.42, 124.70, 124.48, 119.96,119.66, 114.84(2C), 113.61, 112.37, 112.31, 110.42, 107.16, 102.01, 65.08,64.16. ESI-MS m / z: 468.10 [M+H] + .

[0111] Example 28 Synthesis of Gn-3i, a substituted coumarin-benzoxazole derivative

[0112] The difference from Example 1 is that the coumarin intermediate in this example is 7-(3-bromopropoxy)-4-(trifluoromethyl)-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain the substituted coumarin-benzoxazole derivative Gn-3i, yield: 26.3%.

[0113] 1 H NMR (400 MHz, CDCl3) δ 8.23 ​​– 8.15 (m, 2H), 7.77 – 7.71 (m, 1H),7.62 (dq, J = 9.0, 2.0 Hz, 1H), 7.57 – 7.52 (m, 1H), 7.36 – 7.29 (m, 2H), 7.07 – 7.02 (m, 2H), 6.96 – 6.88 (m, 2H), 6.61 (s, 1H), 4.27 (td, J = 6.0, 4.6 Hz, 4H), 2.36 (p, J = 6.0 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 163.06,162.71, 161.42, 159.40, 156.32, 150.68, 142.23, 141.73, 129.45(2C), 126.42,124.70, 124.48, 119.96, 119.66, 114.84(2C), 113.61, 112.37, 112.31, 110.42,107.16, 102.01, 65.08, 64.16, 28.91. ESI-MS m / z: 482.11 [M+H] + .

[0114] Example 29 Synthesis of Gn-4i, a substituted coumarin-benzoxazole derivative

[0115] The difference from Example 1 is that the coumarin intermediate in this example is 7-(4-bromobutoxy)-4-(trifluoromethyl)-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain the substituted coumarin-benzoxazole derivative Gn-4i, yield: 58.5%.

[0116] 1 H NMR (600 MHz, DMSO- d 6 )) δ 8.13 (d, J = 8.4 Hz, 2H), 7.76 (q, J =4.2 Hz, 2H), 7.62 (d, J = 8.8 Hz, 1H), 7.40 – 7.37 (m, 2H), 7.17 (d, J = 8.5Hz, 3H), 7.08 (d, J = 9.3 Hz, 1H), 6.85 (s, 1H), 4.22 (s, 2H), 4.18 (s, 2H), 1.94 (s, 4H). 13 C NMR (101 MHz, CDCl3) δ 163.18, 162.99, 161.75, 159.48,156.36, 150.67, 142.26, 141.77, 129.41(2C), 126.33 (d,J = 2.5 Hz), 124.63,124.45, 120.25, 119.62, 114.82(2C), 113.73, 112.16, 112.11, 110.40, 106.95,101.83, 68.56, 67.84, 28.86, 28.67. ESI-MS m / z: 496.13 [M+H] + .

[0117] Example 30 Synthesis of Gn-5i, a substituted coumarin-benzoxazole derivative

[0118] The difference from Example 1 is that the coumarin intermediate in this example is 7-(5-bromopentoxy)-4-(trifluoromethyl)-2 H -Crone-2-one replaced by 7-(2-bromoethoxy)-2 H -Crotene-2-one, other parameters and operations are as described in Example 1, to obtain the substituted coumarin-benzoxazole derivative Gn-5i, yield: 25.4%.

[0119] 1 H NMR (400 MHz, CDCl3) δ 8.22 – 8.15 (m, 2H), 7.76 – 7.70 (m, 1H),7.61 (dq, J = 9.0, 2.0 Hz, 1H), 7.57 – 7.52 (m, 1H), 7.35 – 7.30 (m, 2H), 7.04 – 7.00 (m, 2H), 6.91 (dd, J = 9.0, 2.5 Hz, 1H), 6.86 (d, J = 2.5 Hz,1H), 6.60 (s, 1H), 4.08 (td, J = 6.3, 3.8 Hz, 4H), 1.98 – 1.86 (m, 4H), 1.72(dq, J = 8.4, 3.5 Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ 163.18, 162.99, 161.75,159.48, 156.36, 150.67, 142.26, 141.77, 129.41(2C), 126.33 (d, J= 2.5 Hz),124.63, 124.45, 120.25, 119.62, 114.82(2C), 113.73, 112.16, 112.11, 110.40,106.95, 101.83, 68.56, 67.84, 28.86, 28.67, 22.69. ESI-MS m / z: 510.15 [M+H] + .

[0120] Experimental Example 1: Inhibitory effect of substituted coumarin-benzoxazole derivatives on monoamine oxidase-B This experiment aimed to determine the inhibitory activity of the substituted coumarin-benzoxazole derivatives obtained in Examples 1-30 against monoamine oxidase-B (MAO-B) using fluorescence spectrophotometry. Results are expressed as inhibition rates, with ladostigil as a positive control. All tests were performed on a PowerWave XS2 full-wavelength microplate reader, with absorbance measured at 490 nm. The compound concentration was 40 μM, and the inhibition rate was calculated using the following formula: Inhibition rate (%) = [1 - (sample - sample background) / (blank group - blank background)] × 100%. In the blank group, 10 µL of PBS (pH=7.6) replaced 10 µL of sample solution; in the blank background group, 30 µL of PBS (pH=7.6) replaced 30 µL of substrate; in the sample background group, 10 µL of PBS (pH=7.6) replaced 10 µL of sample solution; and in the sample background group, 30 µL of PBS (pH=7.6) replaced 30 µL of substrate.

[0121] (1) Preparation of sample solution: Weigh out the samples separately and dissolve them in dimethyl sulfoxide (DMSO) to prepare a 10 mM concentration. Store the DMSO in a -20 ℃ freezer. Before use, dilute the DMSO with phosphate buffer (0.2 mol / L, pH 7.6) to the required concentration so that the final concentration of DMSO is less than or equal to 0.5% (v / v).

[0122] (2) Preparation of enzyme stock solution: Monoamine oxidase B was purchased from Sigma; a certain amount of monoamine oxidase B was weighed and diluted with deionized water to the appropriate activity range.

[0123] (3) Preparation of substrate stock solution: Tyramine was purchased from Sigma-Aldrich. A certain amount of tyramine was weighed and prepared into a 2.5 mM solution with phosphate buffer (0.2 mol / L, pH 7.6), and stored in the dark at 4 ℃.

[0124] (4) Preparation of the colorimetric reagent stock solution: Weigh out a certain amount of vanillic acid, 4-aminoantipyridine and horseradish peroxidase, and prepare a colorimetric solution (1 mM vanillic acid, 0.5 mM 4-aminoantipyridine and 4 U / mL horseradish peroxidase) with phosphate buffer (0.2 mol / L, pH 7.6), and store it in the dark at 4 ℃.

[0125] (5) Test: In a 96-well plate, 10 μL of enzyme solution and 10 μL of sample solution were added respectively. The plate was incubated at 37 °C for 20 min. Immediately after incubation, 30 μL of substrate and 10 μL of chromogenic solution were added. The plate was incubated at 37 °C for 60 min. The absorbance was measured at λ = 490 nm using a microplate reader. The experimental results are shown in Table 1.

[0126] Table 1. Inhibitory activity of substituted coumarin-benzoxazole derivatives against MAO-B

[0127] As shown in Table 1, all compounds obtained in this invention exhibit varying degrees of inhibitory activity against MAO-B. Among them, compound Gn-5d showed the strongest inhibitory activity, reaching 73.6%, with activity even stronger than the positive control ladostigil. This demonstrates that the substituted coumarin-benzoxazole derivatives obtained in this invention can be used to prepare anti-Alzheimer's drugs based on MAO-B inhibition.

[0128] Additionally, based on the compound structures and the overall structure-activity relationship in Table 1, the following may be observed: benzoxazole linkers attached to the 4-position of the coumarin core exhibit better activity than those attached to the 7-position; compounds with methyl substitutions at the 3- and 4-positions of the aromatic ring in the coumarin moiety show better activity; introducing electron-donating groups at the 3- and 4-positions of the coumarin core significantly enhances inhibitory activity; the chain length between the coumarin and benzoxazole moieties has a significant impact on activity, with a carbon chain length of 5 being the most favorable.

[0129] Experimental Example 2: In vitro antioxidant activity experiment of substituted coumarin-benzoxazole derivatives This experimental example aims to determine the in vitro antioxidant activity of the substituted coumarin-benzoxazole derivatives obtained in Examples 1-30 using the ORAC method. AAPH was used as the source of peroxide free radicals, and sodium fluorescein (FL) was used as a fluorescent indicator to evaluate the antioxidant capacity of some compounds. The experimental results are expressed as Trolox equivalents. The specific steps include: (1) Preparation of phosphate buffer (PBS): Take an appropriate amount of phosphate and dilute it with ultrapure water to obtain a 75 mM phosphate solution; weigh 8.56 g of dipotassium hydrogen phosphate and dissolve it in 500 mL of ultrapure water, and adjust the pH to 7.4 with phosphate solution to obtain a 75 mM phosphate buffer with pH 7.4.

[0130] (2) AAPH solution (prepare fresh): Accurately weigh 0.0588 g of AAPH, dissolve and dilute with 5.42 mL of phosphate buffer to prepare an AAPH solution with a concentration of 40.0 mM.

[0131] (3) Preparation of sodium fluorescein solution: Accurately weigh 0.0650 g of sodium fluorescein (FL) and dissolve it in 50 mL of high-purity water to prepare a 3.4 mM FL solution. Store it in a refrigerator at 4 ℃. When using, take 2 μL of the above solution and dissolve it in 50 mL of phosphate buffer solution to obtain a 136 nM FL solution.

[0132] (4) Preparation of Trolox solution: Accurately weigh 2.50 mg of Trolox and dissolve it in 1000 μL of DMSO using a pipette to obtain a 10 mM Trolox solution; during the experiment, accurately pipette the Trolox DMSO solution and dilute it with phosphate buffer to the test concentration.

[0133] (5) Preparation of compound solution: Weigh an appropriate amount of the compound accurately using a precision analytical balance, dilute it with DMSO to a 1 mM transparent solution, and dilute it with phosphate buffer to the required concentration before use.

[0134] (6) Antioxidant activity test: 20 μL of different concentrations of the compound or Trolox, and 120 μL of FL dilution buffer were respectively pipetted into black 96-well culture plates, mixed with a pipette, and incubated at 37 ℃ for 15 min. Then, 60 μL of AAPH was quickly added. Fluorescence values ​​were measured and recorded every 1 min using a multi-mode microplate reader. The excitation wavelength was 485 nm, and the emission wavelength was 535 nm, for a total of 240 min. A blank control was prepared using 20 μL of PBS instead of the compound. The area under the curve (AUC) was calculated using ORIGIN software. The formula for calculating the protective area of ​​the sample was: Net AUC = AUC antioxidant – AUCblank. The ORAC-FL value was calculated as: [(AUC Sample - AUC blank) / (AUC Trolox - AUC blank)] / [Trolox concentration / sample concentration). The sample ORAC value was expressed as a Trolox equivalent.

[0135] Table 2. In vitro antioxidant activity of substituted coumarin-benzoxazole derivatives

[0136] As shown in Table 2, some of the compounds obtained in this invention exhibit good antioxidant activity in vitro. Among them, compounds Gn-5b, Gn-4d, and Gn-5d have ORAC values ​​of 3.8, 3.9, and 3.6 at a concentration of 5 μM, respectively, demonstrating antioxidant activity comparable to curcumin. This proves that the substituted coumarin-benzoxazole derivatives obtained in this invention can be used to prepare drugs for treating Alzheimer's disease based on their antioxidant properties.

[0137] In addition, some structure-activity relationships can be seen from the compound structures and the data in Table 2: the introduction of electron-donating groups on the coumarin core results in higher antioxidant activity; the chain length between the coumarin moiety and the benzoxazole moiety has a significant impact on the activity, and the activity is better when the carbon chain length is 4 or 5.

[0138] Experimental Example 3: Inhibition of Aβ by Substituted Coumarin-Benzoxazole Derivatives 1-42 Self-aggregation This experimental example aims to determine the inhibition of Aβ by the substituted coumarin-benzoxazole derivatives obtained in Examples 1-30 using the ThT method. 1-42 Self-aggregation activity, compounds and Aβ 1-42 The final concentration was 20 μM. Curcumin and 7-hydroxycoumarin were used as positive controls. The specific steps included: 1. Solution preparation: (1) 20 mM pH 7.4 phosphate buffer solution (PBS): Weigh 3.618 g Na2HPO4 and 0.6027 g KH2PO4, add 100 mL of ultrapure water, and after the solids have completely dissolved, make up to 200 mL with ultrapure water and adjust the pH of the solution to 7.4.

[0139] (2) Aβ 1-42 Protein solution: Dissolve 1 mg of protein in 100 μL of 1% NH4OH solution to obtain a concentration of 2300 μM. Store at -80°C for later use. Dilute with PBS buffer to 40 μM before use.

[0140] (3) 50 mM glycine-NaOH buffer: Weigh 0.938 g glycine, dissolve it in 250 mL ultrapure water, adjust the pH to 8.50 with 1 mol / L NaOH solution, and store at 4℃.

[0141] (4) 5 μM thiamine T solution (prepared fresh): Weigh 2.2 mg of thiamine T powder and dissolve it in 689 μL of glycine-NaOH buffer solution at pH 8.5. Sonicate the solid until it is completely dissolved and store in the dark.

[0142] (5) Preparation of compound solution: Weigh an appropriate amount of the compound accurately using a precision analytical balance, dilute it with DMSO to a 10 mM transparent solution, and dilute it with phosphate buffer to the test concentration before use.

[0143] 2. Inhibitory activity test: Take 10 μL of 40 μM Aβ respectively 1-42 The protein was mixed with 10 μL of a 40 μM compound and incubated at 37°C for 48 h. The blank control was 10 μL of 40 μM Aβ. 1-42 The protein was mixed with 10 μL of pH 7.4 phosphate buffer and incubated together; the positive control was Aβ. 1-42 The protein was co-incubated with resveratrol. After 72 h, the incubation solution was transferred to black 96-well plates, and 180 μL of 5 μM thiamine T solution was added. The plates were then incubated in the dark at room temperature for 5 min. Finally, the fluorescence absorbance was measured using a multi-mode microplate reader, with an excitation wavelength of 450 nm and an absorption wavelength of 485 nm. The Aβ value in the negative control experiment was used as a reference. 1-42 Using the fluorescence intensity of the compound bound to thiosulfate T as a control, the effect of the compound on Aβ was determined. 1-42 Inhibition rate of protein aggregation. The results are shown in Table 3.

[0144] The results show that the compound described in this invention has an effect on Aβ. 1-42 These compounds exhibit strong inhibitory activity, with most showing better activity than curcumin. Compounds Gn-4d and Gn-5d demonstrated the strongest inhibitory activity, reaching 96.2% and 94.1%, respectively. This demonstrates that the substituted coumarin-benzoxazole derivatives described in this invention have significant development potential and can be used to inhibit Aβ. 1-42 Self-aggregation, used to prepare drugs for treating Alzheimer's disease.

[0145] Additionally, based on the compound structure and the overall structure-activity relationship in Table 3, the following may be observed: electron-donating groups on the aromatic ring of the coumarin moiety have higher activity than electron-withdrawing groups; the benzoxazole linker connected at position 7 of the coumarin core has better activity than position 4; furthermore, the chain length between the coumarin moiety and the benzoxazole has an effect on the activity, with a carbon chain length of 4 being more desirable.

[0146] Table 3. Aβ of substituted coumarin-benzoxazole derivatives 1-42 Self-aggregation inhibition activity

[0147] Experimental Example 4: Inhibitory effect of substituted coumarin-eugenol derivatives on butyrylcholinesterase This experiment aimed to determine the inhibitory effect of the substituted coumarin-benzoxazole derivatives obtained in Examples 1-30 on butyrylcholinesterase using the Ellman assay. Results were expressed as inhibition rates, with Tacrine used as a positive control. All tests were performed on a PowerWave XS2 full-wavelength microplate reader at 37°C. Data analysis was performed using Origin software. The specific steps are as follows: (1) Preparation of drug solution: Weigh a certain amount of each sample to be analyzed and dissolve it in dimethyl sulfoxide (DMSO) to prepare a 10 mM concentration. Store it in a -20°C freezer. Before use, dilute it with phosphate buffer (0.1 mol / L, pH 8.0) to the required concentration so that the final concentration of DMSO is less than or equal to 0.5% (v / v).

[0148] (2) Preparation of enzyme stock solution: Butyrylcholinesterase (EC 3.1.1.7, from electric ell.) was purchased from Sigma-Aldrich; a certain amount of butyrylcholinesterase was weighed and diluted with deionized water to the appropriate activity range.

[0149] (3) Preparation of substrate stock solution: Acetylthiocholine (ATC) was purchased from Sigma-Aldrich. A certain amount of ATC was weighed and prepared into a 0.01 mol / L solution with phosphate buffer (0.1 mol / L, pH 8.0), and stored in the dark at 4°C.

[0150] (4) Preparation of the colorimetric reagent stock solution: The colorimetric reagent 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) was purchased from Sigma-Aldrich. A certain amount of DTNB was weighed and prepared into a 0.01 mol / L solution with phosphate buffer (0.1 mol / L, pH 8.0), and stored in the dark at 4°C.

[0151] (5) Test: In a 96-well plate, six wells were selected, and 10 μL of enzyme solution and 0, 5, 10, 20, 35, and 50 μL of the test compound solution were added respectively. 0.1 mol / L pH 8.0 phosphate buffer was added to bring the total volume to 100 μL. The plate was incubated at 37 ℃ for 15 min using a full-wavelength microplate reader. Immediately afterwards, a mixture of 10 μL ATC solution, 10 μL DTNB solution, and 80 μL phosphate buffer was added to a total volume of 100 μL. The absorbance was measured at λ=412 nm for 2 min. The experimental results are shown in Table 4. The results indicate that some of the compounds described in this invention have moderate to high inhibitory activity. Among them, compounds Gn-4d and Gn-5d showed the best inhibition of butyrylcholinesterase activity, with inhibition rates of 64.3% and 67.8%, respectively, and can be used to prepare drugs for treating Alzheimer's disease.

[0152] Table 4. Inhibitory activity of substituted coumarin-benzoxazole derivatives against butyrylcholinesterase

[0153] Experimental Example 5: Toxicity Study of Substituted Coumarin-Benzoxazole Derivatives on Nerve Cells This experimental example aims to determine the toxicity of the substituted coumarin-benzoxazole derivatives obtained in Examples 1-30 to nerve cells (SH-SY5Y) using the MTT assay. The specific steps are as follows: 1. Solution preparation (1) DMEM medium: Dissolve the dry powder medium in 300 mL of ultrapure water in a 1000 mL beaker, then rinse the inside of the packaging twice with 300 mL of ultrapure water, combine the solutions, and stir magnetically to dissolve completely; add 3.7 g sodium bicarbonate and 2.38 g HEPES, and stir magnetically to dissolve completely; adjust the pH to 7.5 with 10 M sodium hydroxide while stirring, filter sterilize with a 0.22 μM filter membrane in a clean bench, and store in a refrigerator at 4 ℃; add antibiotics (final concentration of penicillin 100 U / mL, streptomycin 100 μg / mL) and fetal bovine serum (10%) when using.

[0154] (2) PBS buffer solution: accurately weigh 8 g HCl, 0.2 g KH2PO4 and 2.88 g H2HPO4•12H2O, dissolve in ultrapure water and bring to a final volume of 1 L, autoclave at 120 ℃ for 20 min, and store in a refrigerator at 4 ℃.

[0155] (3) MTT solution: Prepare MTT to 5 g / L with PBS solution, filter it with a 0.22 μM filter membrane for sterilization, and store it in a refrigerator at 4 ℃ protected from light.

[0156] 2. Culture of SH-SY5Y nerve cells The neural cell line SH-SY5Y was cultured in DMEM medium in a conventional incubator at 37 ℃, saturated humidity, and an environment of 5% CO2 and 95% air, and passaged every 2-3 days.

[0157] 3. Neurotoxicity assay (1) Take cells in the logarithmic growth phase, digest them with 0.25% trypsin, wash them twice with PBS, resuspend them in DMEM medium, count them under a microscope using a cell counting chamber, and adjust the cell concentration to 5 × 10⁶ cells / year. 4 Cells were seeded at 100 μL / well in a 96-well cell culture plate and cultured for 24 h to allow the cells to adhere.

[0158] (2) Remove the original culture medium and add compound solutions of different concentrations diluted with DMEM culture medium, 100 μL per well, with 5 replicates. Add culture medium to the blank and control groups instead of the compound, and incubate at 37 ℃ and 5% CO2 for 48 h. Add culture medium containing 5 mg / mL MTT to the sample group and control group 100 μL / well 4 h before the end of the experiment, and continue incubation for 4 h.

[0159] (3) Discard the supernatant, add 100 μL of DMSO to each well, shake to fully dissolve the product formazan, and measure the absorbance (OD value) of each well on a full-wavelength microplate reader at a wavelength of 570 nm; cell viability (%) in each sample = (OD sample - OD blank) / (OD control - OD blank) × 100%; cell inhibition rate (%) in each sample = 100% - cell viability (%) in each sample. Plot the inhibition rate against the concentration. The concentration with an inhibition rate of 50% is the IC50 of the compound. 50 Values. See Table 5 for the results.

[0160] Table 5. Toxicity of substituted coumarin-benzoxazole derivatives on nerve cells (SH-SY5Y)

[0161] As shown in Table 5, the partially substituted coumarin-benzoxazole derivatives obtained in this invention have an effect on the IC50 of SH-SY5Y cells. 50 Values ​​greater than 90 μM indicate low neurotoxicity; among them, the compound Gn-5d, which exhibits the strongest inhibitory activity against MAO-B, has an IC50 value greater than 90 μM. 50 The value is 117.6 μM, indicating good safety.

[0162] Experimental Example 6: Effects of coumarin-benzoxazole derivatives Gn-4d and Gn-5d on Aβ 1-42 Study on the neuroprotective effect of induced SH-SY5Y cell damage This experimental example aims to determine the effect of the substituted coumarin-benzoxazole derivatives Gn-4d and Gn-5d obtained in Examples 14 and 15 on Aβ using the MTT assay. 1-42 The neuroprotective effect of induced SH-SY5Y cell damage is determined through the following steps: SH-SY5Y cells were passaged in 96-well plates at a cell density of 1 × 10⁶ cells per well. 4 After removing the culture medium, Aβ was added separately. 1-42 Cells were incubated with (20 μM) solution and 10 μM of different compounds (Gn-4d and Gn-5d) for 24 h, with 7-hydroxycoumarin as a positive control. Cell viability was determined by the MTT assay as described in Example 5 above. The results are shown below. Figure 1 .

[0163] Depend on Figure 1 It is evident that the substituted coumarin-benzoxazole derivatives Gn-4d and Gn-5d obtained in this invention have a positive effect on Aβ. 1-42 The induced SH-SY5Y cell damage all showed good neuroprotective effects and exhibited better protective ability than 7-hydroxycoumarin; among them, the cell survival rate of SH-SY5Y cells increased to 92.4% after treatment with 20 μM compound Gn-5d.

[0164] Example 7: Study on the effects of coumarin-benzoxazole derivative Gn-5d on scopolamine-induced memory impairment in mice. This experiment aims to evaluate the effects of the coumarin-benzoxazole derivative Gn-5d obtained in Example 15 on scopolamine-induced memory impairment in mice using a passive avoidance test. The specific steps are as follows: (1) Establishment of AD mouse model Sixty male SD mice (3 months old) were used and placed in an environment with a temperature of 22-25 ℃, relative humidity of 50-70%, and a light-dark cycle of 12 h. The 60 male SD mice were randomly divided into 6 groups: a) control group (injected with physiological saline and gavaged with distilled water); b) AD model group (scopolamine 3.0 mg / kg); c) positive control group (donepezil, 8.0 mg / kg); d) high-dose group (IM5-H 8.0 mg / kg); e) medium-dose group (Gn-5d-M 4.0 mg / kg); f) low-dose group (Gn-5d-L 2.0 mg / kg).

[0165] (2) Passive avoidance experiment In vivo memory enhancement was assessed using a passive avoidance experiment in mice, comprising two separate tests (a training test and a test 24 hours later). The experimental setup consisted of two identical compartments (a light compartment and a dark compartment) separated by a guillotine door, illuminated by 250 lx LED lights. In the training test, mice were first placed in the light chamber and allowed free movement for 5 minutes to familiarize themselves with their environment. The door was then opened, and the mice quickly entered the dark compartment while an electrical stimulator was activated, delivering an electric shock (24V, 0.5mA) to the animal's paws. This training was repeated for 5 minutes. Mice that failed to enter the dark compartment within 180 seconds were eliminated and subjected to the test.

[0166] Mice were administered Gn-5d (2.0, 4.0, and 8.0 mg / kg) or donepezil (8.0 mg / kg) by gavage one hour before each training session. Thirty minutes later, scopolamine (3.0 mg / kg) was injected intraperitoneally to induce memory impairment. The training was conducted 24 hours later. Mice were placed back into the light chamber and the door was opened. The total test time was set to 5 minutes. Delay time and the number of errors were recorded during the test. Delay time (latency) was the time it took for the mouse to enter the dark chamber, and the number of errors was the number of times the mouse entered the dark chamber within 5 minutes. Results are shown below. Figure 2 .

[0167] Depend on Figure 2 As shown, the latency in the scopolamine-treated group (model group, 95 sec) was significantly shorter than that in the control group (228 sec), and the average number of errors within 5 minutes was significantly increased (5.1 in the model group, 1.9 in the control group), indicating that scopolamine induces memory impairment in mice. Treatment with donepezil and different concentrations of compound Gn-5d (2.0, 4.0, 8.0 mg / kg) significantly reversed both latency and average number of errors compared to the model group. Furthermore, compound Gn-5d prolonged latency and reduced the number of errors in a dose-dependent manner. Specifically, the 8.0 mg / kg dose of compound Gn-5d showed better efficacy in increasing latency and reducing the number of errors compared to the donepezil group.

[0168] As can be seen from the above, the substituted coumarin-benzoxazole derivative obtained in this invention has good MAO-B inhibitory activity and Aβ inhibitory activity. 1-42 It exhibits aggregation and antioxidant activity, and has low toxicity to nerve cells, demonstrating high safety. Among these, compound Gn-5d exhibits activity against Aβ. 1-42 The induced SH-SY5Y cell damage exhibits strong neuroprotective effects; animal experiments have shown that high-dose compound Gn-5d can effectively reverse scopolamine-induced memory impairment in mice. Therefore, the compound provided by this invention is highly suitable for preparing drugs against Alzheimer's disease.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A substituted coumarin-benzoxazole derivative, characterized in that, It has the structure shown in formula (I) or (II): In formulas (I) and (II), R1 is selected from one or more of hydrogen, methyl, chloro, and formyloxyethyl; R2 is selected from hydrogen, methyl, trifluoromethyl, and benzene ring; and n is a positive integer from 2 to 5.

2. The substituted coumarin-benzoxazole derivative according to claim 2, characterized in that, The substituted coumarin-benzoxazole derivative has any of the following structures: 。 3. A method for preparing the substituted coumarin-benzoxazole derivative as described in any one of claims 1-2, characterized in that, The synthesis route is as follows: ， Specifically, the following steps are included: S1. Reacting a compound of formula (III) or (V) with Br-(CH2) n -Br undergoes a substitution reaction in an alkaline environment to give compounds of formula (IV) or (VI); S2. The compound of formula (IV) or (VI) obtained in step S1 is subjected to a substitution reaction with 4-(benzo[d]oxazol-2-yl)phenol under alkaline conditions to obtain compound (I) or compound (II). Wherein, R1 is selected from one or more of hydrogen, methyl, chloro, and formyloxyethyl; R2 is selected from hydrogen, methyl, trifluoromethyl, and benzene ring; n is a positive integer from 2 to 5.

4. The preparation method according to claim 3, characterized in that, The alkaline environment in step S1 is formed by one or more of cesium carbonate, potassium carbonate, and sodium carbonate, with a reaction temperature of 25–60°C and a reaction time of 2–8 h. The alkaline environment in step S2 is formed by one or more of cesium carbonate and potassium carbonate, with one or more of N,N-dimethylformamide and acetonitrile as the solvent, with a reaction temperature of 25–100°C and a reaction time of 2–8 h. The reaction is then purified by column chromatography.

5. The use of the substituted coumarin-benzoxazole derivative as described in any one of claims 1-2 in the preparation of monoamine oxidase-B inhibitors.

6. The use of the substituted coumarin-benzoxazole derivative as described in any one of claims 1-2 in the preparation of butyrylcholinesterase inhibitors.

7. The use of the substituted coumarin-benzoxazole derivative as described in any one of claims 1-2 in the preparation of Aβ aggregation inhibitors.

8. The use of the substituted coumarin-benzoxazole derivative as described in any one of claims 1-2 in the preparation of antioxidants.

9. The use of the substituted coumarin-benzoxazole derivative as described in any one of claims 1-2 in the preparation of a medicament for treating Alzheimer's disease, vascular dementia, myasthenia gravis, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis.

10. A pharmaceutical preparation, characterized in that, The product comprises the substituted coumarin-benzoxazole derivative according to any one of claims 1-2, and the dosage form is selected from tablets, pills, capsules, injections, suspensions or emulsions.

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