A novel substituted indigo-coumarin derivative, and a preparation method and application thereof

Abstract

The application relates to the technical field of biological medicine, and discloses a novel substituted indigo-coumarin derivative, a preparation method and application thereof. The novel substituted indigo-coumarin derivative has the effects of significantly inhibiting monoamine oxidase-B activity, inhibiting A beta aggregation, resisting oxidation, inhibiting acetylcholinesterase and metal ion chelation, and has therapeutic effects on diseases related to the above mechanisms; in particular, the novel substituted indigo-coumarin derivative has significant therapeutic effects on multiple therapeutic targets of Alzheimer's disease, has low biological toxicity, high safety, has high medical research and market application values. On the other hand, the novel substituted indigo-coumarin derivative is very suitable for large-scale industrial production due to low raw material cost, few reaction steps and simple preparation method.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a novel substituted indigo-coumarin derivative, a preparation method and application thereof. BACKGROUND

[0002] Alzheimer's disease (AD) is a chronic progressive central nervous system degenerative disease, commonly known as "senile dementia".

[0003] Current research shows that the main pathological features of AD patients are the aggregation of β-amyloid protein (Aβ) into senile plaques, the abnormal aggregation of intracellular Tau protein into neurofibrillary tangles (NFT) and the death of neurons. However, so far, the cause of AD has not been clear, and many factors are involved in the pathogenesis; during the research process, many related hypotheses have been proposed to explain the occurrence and development of the disease, including cholinergic hypothesis, amyloid cascade hypothesis, Tau protein hypothesis, oxidative stress hypothesis, metal ion hypothesis, and neuroinflammation hypothesis. And for these hypotheses, corresponding solutions have also been proposed. First, the amyloid cascade hypothesis, aiming at the metabolic process of Aβ, researchers have proposed various treatment strategies. One of them is to design and synthesize small molecule ligands that can inhibit the aggregation of Aβ. Secondly, the cholinergic hypothesis, which believes that the decrease of cholinergic nerve function is the main cause of AD. According to this, scientists have developed cholinesterase inhibitors (ChEI). The third is the oxidative stress hypothesis, which believes that abnormal metabolism of substances and energy in brain tissue leads to accumulation of free radicals, causing oxidative damage, cell apoptosis and AD characteristic neuropathological changes. In this regard, researchers have proposed to design and synthesize compounds with antioxidant capacity to solve the problem of oxidative stress. So far, this strategy has achieved good results. In addition, studies have shown that some metal ions such as copper, iron, zinc, etc. can bind to Aβ and promote its aggregation process. Electron microscopy results show that the concentration of copper and zinc ions in senile plaques is twice that of normal people, and metal ions such as copper and iron can promote the generation of ROS, leading to an increase in oxidative stress, so metal ion chelators have become a means of treating AD.

[0004] A large number of experiments have shown that the activity of MAO-B gradually increases with age, especially in the surrounding of senile plaques in AD patients, and the increase of its activity can lead to an increase in free radicals with neurotoxicity in the brain, an increase in oxidative stress, and at the same time further accelerate the aggregation of Aβ protein and the over-phosphorylation of tau protein in the brain, ultimately causing nerve damage leading to neuronal death, therefore, inhibitors of MAO-B can be used to treat AD in recent years.

[0005] From the pathogenesis of AD, the pathogenesis of AD is multiple, and each mechanism is closely related and interacts with each other. Therefore, the drug treatment effect of single target is not ideal. More and more studies show that the drug acting on multiple targets related to the disease, i.e. multi-target drug, may have better treatment effect. The design of multi-target drug is to obtain the required multiple biological activities by using biological structure information and pharmacophore model. The characteristics of multi-target drug action are to play a role in different pathological and physiological links of the disease and enhance the curative effect. Therefore, designing and synthesizing multi-target drugs with anti-Aβ aggregation activity, metal ion complexing, MAO-B inhibiting activity and antioxidant activity may be an effective strategy for treating AD. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the single target drug for treating AD, and to provide a novel substituted indigo-coumarin derivative with multi-target treatment effect for AD, and a preparation method and application thereof.

[0007] The novel substituted indigo-coumarin derivative provided by the present application has the structure of formula (I):

[0008]

[0009] In the formula, R is selected from one or more of hydrogen, halogen and methoxy; R1 is selected from hydrogen or methyl, and n is a positive integer of 1-4.

[0010] Preferably, R is selected from one or more of hydrogen, 5-halogen, 5-methoxy, 6-halogen; R1 is selected from hydrogen or methyl, and n is a positive integer of 1-4.

[0011] Therefore, the present application claims the application of the novel substituted indigo-coumarin derivative in the preparation of monoamine oxidase-B inhibitors.

[0012] In addition, the present application also claims the application of the novel substituted indigo-coumarin derivative in the preparation of acetylcholinesterase inhibitors.

[0013] In addition, the present application also claims the application of the novel substituted indigo-coumarin derivative in the preparation of Aβ aggregation inhibitors.

[0014] In addition, the present application also claims the application of the novel substituted indigo-coumarin derivative in the preparation of antioxidants.

[0015] In addition, the present application claims the application of the novel substituted indigo-coumarin derivative in the preparation of metal complexing agents.

[0016] Based on the therapeutic principles of inhibiting monoamine oxidase-B, inhibiting acetylcholinesterase, anti-Aβ aggregation, metal ion chelation and anti-oxidation, the novel substituted indigo-coumarin derivative provided by the application has therapeutic effects on diseases related to the above mechanisms.

[0017] Therefore, the application also claims the use of the novel substituted indigo-coumarin derivative in the preparation of a medicament for treating Alzheimer's disease, cerebral vascular dementia, myasthenia gravis, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis.

[0018] Further, the dosage form of the pharmaceutical preparation is a tablet, a pill, a capsule, an injection, a suspension or an emulsion.

[0019] Compared with the related art, the novel substituted indigo-coumarin derivative provided by the application has the following beneficial effects:

[0020] The application provides a novel substituted indigo-coumarin derivative, which has significant effects of inhibiting Aβ aggregation, inhibiting monoamine oxidase-B activity, inhibiting acetylcholinesterase activity, anti-oxidation and metal ion chelation, and has therapeutic effects on diseases related to the above mechanisms; in particular, the novel substituted indigo-coumarin derivative has significant therapeutic effects on multiple therapeutic targets of Alzheimer's disease, has low biological toxicity, high safety, and has high medical research and market application values. On the other hand, the novel substituted indigo-coumarin derivative has low raw material cost, few reaction steps, a simple preparation method, and is very suitable for large-scale industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 FIG. 4 is an ultraviolet spectrum diagram of the interaction between the substituted indigo-coumarin derivative IC-4g in the experimental example 24 of the application and metal ions;

[0022] Figure 2 FIG. 6 is a result diagram of the neuroprotective effect of the compound on Aβ-induced SH-SY5Y cell damage determined by the MTT method in the embodiment 7;

[0023] Figure 3 FIG. 8 is a result diagram of the passive avoidance experiment in the embodiment 8. DETAILED DESCRIPTION

[0024] The application will be further described below in combination with the drawings and specific embodiments.

[0025] Embodiment 1: Synthesis of the substituted indigo-coumarin derivative IC-2a

[0026]

[0027] In a 25 mL round bottom flask, 5 mL of DMF, 7-hydroxycoumarin (0.15 g, 0.93 mmol) and anhydrous potassium carbonate (0.24 g, 1.76 mmol) were added, and the reaction was stirred at room temperature for 30 min, then indigo intermediate N-(2-bromoethyl)-5-chloroindigo (0.25 g, 0.88 mmol) was added, and the reaction was continued to stir at 60 °C, and the reaction progress was monitored by TLC; after the reaction was completed, the reaction solution was poured into 40 mL of ice water, stirred at room temperature, and the solution pH was adjusted to about 7-8 with dilute hydrochloric acid, and then continue to stir for 30 min, so that the solid is fully precipitated, then suction filtration, the obtained solid was air dried, and the substituted indigo-coumarin derivative IC-2a was separated by silica gel column chromatography, yield: 39.1%.

[0028] 1 H NMR (400 MHz, CDC13) δ 7.64-7.55 (m, 3H), 7.36 (d, J = 9.3 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 6.7 Hz, 2H), 6.27 (d, J = 9.5 Hz, 1H), 4.32 (t, J = 4.9 Hz, 2H), 4.18 (t, J = 4.9 Hz, 2H). 13 C NMR (101 MHz, CDC13) δ 182.13, 161.24, 161.08, 157.95, 155.68, 149.31, 143.12, 137.87, 129.84, 128.99, 125.63, 118.41, 113.65, 113.12, 112.32, 111.13, 101.73, 67.65, 40.34. ESI-MS m / z: 370.8 [M+H] + .

[0029] Synthesis of substituted indigo-coumarin derivative IC-3a of example 2

[0030]

[0031] The difference between this example and example 1 is that the indigo intermediate of this example is replaced by N-(3-bromopropyl)-5-chloroindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations refer to example 1, to obtain substituted indigo-coumarin derivative IC-3a, yield: 42.8%.

[0032] 1H NMR (400 MHz, CDC13) δ 7.63 (d, J = 9.5 Hz, 1H), 7.58 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.78 (d, J = 7.9 Hz, 2H), 6.27 (d, J = 9.5 Hz, 1H), 4.11 (t, J = 5.5 Hz, 2H), 3.97 (t, J = 6.8 Hz, 2H), 2.24 (dd, J = 12.1, 6.1 Hz, 2H). 13 CNMR (101 MHz, CDC13) δ 182.20, 161.41, 161.00, 157.82, 155.83, 149.10, 143.26, 137.76, 129.74, 128.98, 125.53, 118.45, 113.55, 112.99, 112.48, 111.21, 101.56, 65.53, 37.55, 26.96. ESI-MS m / z: 384.5 [M+H] + .

[0033] Synthesis of substituted indigo-coumarin derivative IC-4a

[0034]

[0035] The difference between this example and Example 1 is that N-(4-bromobutyl)-5- chloroindigo is used instead of N-(2-bromoethyl)-5-chloroindigo for the indigo intermediate of this example, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-4a, yield: 52.1%.

[0036] 1 H NMR (400 MHz, CDC13) δ 7.63 (d, J = 9.5 Hz, 1H), 7.58 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.78 (d, J = 7.9 Hz, 2H), 6.27 (d, J = 9.5 Hz, 1H), 4.11 (t, J = 5.5 Hz, 2H), 3.97 (t, J = 6.8 Hz, 2H), 2.24 (dd, J = 12.1, 6.1 Hz, 2H). 13C NMR (126 MHz, CDC13) δ 182.41, 162.11, 160.95, 157.71, 155.89, 149.11, 143.42, 137.70, 129.62, 128.79, 125.50, 118.45, 113.12, 112.88, 112.55, 111.33, 101.32, 68.02, 40.19, 28.50, 26.89, 23.39. ESI-MS m / z: 412.5 [M+H] + .

[0037] Synthesis of substituted indigo-coumarin derivative IC-5a

[0038]

[0039] The difference between this example and Example 1 is that N-(5-bromopentyl)-5- chloroindigo is used instead of N-(2-bromoethyl)-5-chloroindigo for the indigo intermediate of this example, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-5a, yield: 46.8%.

[0040] 1 H NMR (500 MHz, CDC13) δ 7.63 (d, J = 9.5 Hz, 1H), 7.59-7.52 (m, 2H), 7.36 (d, J = 8.6 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.82-6.75 (m, 2H), 6.25 (d, J = 9.5 Hz, 1H), 4.01 (t, J = 6.2 Hz, 2H), 3.77 (t, J = 7.2 Hz, 2H), 1.91-1.85 (m, 2H), 1.83-1.76 (m, 2H), 1.62-1.53 (m, 2H). 13 C NMR (126 MHz, CDC13) δ 182.41, 162.11, 160.95, 157.71, 155.89, 149.11, 143.42, 137.70, 129.62, 128.79, 125.50, 118.45, 113.12, 112.88, 112.55, 111.33, 101.32, 68.02, 40.19, 28.50, 26.89, 23.39. ESI-MS m / z: 412.5 [M+H] + .

[0041] Synthesis of substituted indigo-coumarin derivative IC-2b

[0042]

[0043] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced by N-(3-bromopropyl)-indigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-3b, yield: 50.8%.

[0044] 1 HNMR (400 MHz, CDC13) δ 7.67-7.61 (m, 3H), 7.36 (d, J = 6.8 Hz, 1H), 7.16 (t, J = 5.8 Hz, 2H), 6.80-6.72 (m, 2H), 6.26 (d, J = 7.6 Hz, 1H), 4.32 (t, J = 3.9 Hz, 2H), 4.19 (t, J = 3.9 Hz, 2H). 13 C NMR (101 MHz, CDC13) δ 182.87, 160.99, 160.91, 158.49, 155.69, 151.12, 143.21, 138.35, 129.01, 125.53, 124.04, 117.62, 113.72, 113.18, 112.28, 110.81, 101.76, 66.12, 39.81. ESI-MS m / z: 336.4 [M+H] + .

[0045] Synthesis of substituted indigo-coumarin derivative IC-3b

[0046]

[0047] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced by N-(3-bromopropyl)-indigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-3b, yield: 50.8%.

[0048] 1 H NMR (400 MHz, CDC13) δ 7.67-7.61 (m, 3H), 7.36 (d, J = 6.8 Hz, 1H), 7.16 (t, J = 5.8 Hz, 2H), 6.80-6.72 (m, 2H), 6.26 (d, J = 7.6 Hz, 1H), 4.32 (t, J = 3.9 Hz, 2H), 4.19 (t, J = 3.9 Hz, 2H). 13C NMR (101 MHz, CDCI3): δ 161.55, 161.01, 158.35, 155.82, 150.85, 143.25, 138.39, 128.92, 125.61, 123.85, 117.66, 113.47, 112.91, 112.50, 109.92, 101.64, 100.00, 65.66, 37.33, 27.06. ESI-MS m / z: 350.4 [M+H] + .

[0049] Synthesis of substituted indigo-coumarin derivative IC-4b

[0050]

[0051] The difference between this example and Example 1 is that N-(4-bromobutyl)-indigo is used instead of N-(2-bromoethyl)-5-chloroindigo for the indigo intermediate of this example, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-4b, yield: 54.2%.

[0052] 1 H NMR (500 MHz, CDCI3) δ 7.65-7.58 (m, 3H), 7.37 (d, J = 8.6 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.82 (d, J = 8.6 Hz, 1H), 6.78 (s, 1H), 6.26 (d, J = 9.5 Hz, 1H), 4.08 (s, 2H), 3.83 (d, J = 6.3 Hz, 2H), 1.94 (s, 4H). 13 C NMR (126 MHz, CDCI3) δ 183.37, 161.94, 161.19, 158.26, 155.85, 150.76, 143.40, 138.41, 128.86, 125.62, 123.84, 117.63, 113.22, 112.69, 112.66, 110.08, 101.49, 67.60, 39.79, 26.31, 23.99. ESI-MS m / z: 364.4 [M+H] + .

[0053] Synthesis of substituted indigo-coumarin derivative IC-5b

[0054]

[0055] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced by N-(5-bromopentyl)-indigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations refer to Example 1 to obtain the substituted indigo-coumarin derivative IC-5b, yield: 49.4%.

[0056] 1 H NMR (400 MHz, CDC13) δ 7.66-7.57 (m, 3H), 7.36 (d, J = 8.6 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 6.91 (d, J = 7.9 Hz, 1H), 6.82 (dd, J = 8.6, 2.3 Hz, 1H), 6.77 (d, J = 2.3 Hz, 1H), 6.25 (d, J = 9.5 Hz, 1H), 4.02 (t, J = 6.2 Hz, 2H), 3.78 (t, J = 13.5 Hz, 2H), 1.92-1.86 (m, 2H), 1.84-1.77 (m, 2H), 1.63-1.55 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 183.49, 162.15, 161.27, 158.22, 155.88, 150.88, 143.45, 138.37, 128.79, 125.59, 123.76, 117.62, 113.08, 112.88, 112.52, 110.08, 101.35, 68.10, 40.02, 28.54, 26.99, 23.43. ESI-MS m / z: 378.4 [M+H] + .

[0057] Synthesis of substituted indigo-coumarin derivative IC-2c

[0058]

[0059] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced by N-(2-bromoethyl)-5-methoxyindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations refer to Example 1 to obtain the substituted indigo-coumarin derivative IC-2c, yield: 49.6%.

[0060] 1H NMR (400 MHz, CDC13) δ 7.55 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 15.7 Hz, 1H), 7.09 (d, J = 15.7 Hz, 2H), 7.00 (d, J = 7.3 Hz, 1H), 6.68 (s, 2H), 6.19 (d, J = 8.3 Hz, 1H), 4.25 (d, J = 13.1 Hz, 2H), 4.08 (s, 2H), 3.75 (s, 3H). 13 CNMR (101 MHz, CDC13) δ 182.25, 159.98, 159.87, 157.56, 155.60, 154.66, 144.01, 142.18, 127.97, 123.77, 116.97, 112.67, 112.13, 111.26, 110.86, 108.38, 100.71, 65.22, 54.97, 38.79. ESI-MS m / z: 366.4 [M+H] + .

[0061] Synthesis of substituted indigo-coumarin derivative IC-3c

[0062]

[0063] The difference between this example and Example 1 is that N-(3-bromopropyl)-5- methoxyindigo is used instead of N-(2-bromoethyl)-5-chloroindigo for the indigo intermediate of this example, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-3c, yield: 52.8%.

[0064] 1 H NMR (400 MHz, CDC13) δ 7.55 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 15.7 Hz, 1H), 7.09 (d, J = 15.7 Hz, 2H), 7.00 (d, J = 7.3 Hz, 1H), 6.68 (s, 2H), 6.19 (d, J = 8.3 Hz, 1H), 4.25 (d, J = 13.1 Hz, 2H), 4.08 (s, 2H), 3.75 (s, 3H). 13C NMR (126 MHz, CDC13) δ 184.35, 161.56, 161.11, 158.40, 156.56, 155.80, 144.70, 143.35, 128.95, 124.82, 117.90, 113.43, 112.90, 112.52, 110.96, 109.73, 101.61, 65.62, 56.00, 37.30, 27.06. ESI-MS m / z: 380.4 [M+H] + .

[0065] Synthesis of substituted indigo-coumarin derivative IC-4c, Example 11

[0066]

[0067] The difference between this example and Example 1 is that N-(4-bromobutyl)-5- methoxyindigo is used instead of N-(2-bromoethyl)-5-chloroindigo for the indigo intermediate of this example, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-4c, yield: 51.1%.

[0068] 1 H NMR (400 MHz, CDC13) δ 7.64 (d, J = 9.5 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.18 - 7.13 (m, 2H), 6.88 - 6.84 (m, 1H), 6.82 (dd, J = 8.6, 2.3 Hz, 1H), 6.77 (d, J = 2.1 Hz, 1H), 6.25 (d, J = 9.5 Hz, 1H), 4.08 (d, J = 5.1 Hz, 2H), 3.81 (s, 3H), 3.80 - 3.75 (m, 2H), 1.92 (d, J = 3.2 Hz, 4H). 13 C NMR (101 MHz, CDC13) δ 183.78, 161.95, 161.21, 158.34, 156.53, 155.84, 144.59, 143.43, 128.87, 124.73, 118.04, 113.19, 112.69, 112.65, 111.11, 109.75, 101.48, 67.60, 56.01, 39.78, 26.30, 23.97. ESI-MS m / z: 394.4 [M+H] + .

[0069] Synthesis of substituted indigo-coumarin derivative IC-5c, Example 12

[0070]

[0071] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced with N-(5-bromopentyl)-5-methoxyindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-5c, yield: 52.5%.

[0072] 1 H NMR (400 MHz, CDC13) δ 7.64 (d, J = 9.2 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.14 (d, J = 9.3 Hz, 2H), 6.82 (t, J = 8.0 Hz, 2H), 6.77 (s, 1H), 6.25 (d, J = 9.3 Hz, 1H), 4.01 (s, 2H), 3.81 (s, 3H), 3.75 (d, J = 7.2 Hz, 2H), 1.88 (s, 2H), 1.83 - 1.76 (m, 2H), 1.60 (d, J = 6.5 Hz, 2H). 13 CNMR (101 MHz, CDC13) δ 183.41, 162.09, 161.35, 158.27, 156.56, 155.89, 144.34, 143.54, 128.75, 124.74, 118.18, 113.04, 112.74, 112.61, 111.21, 109.88, 101.31, 67.88, 56.14, 40.09, 28.46, 26.48, 23.64. ESI-MS m / z: 408.4 [M+H] + .

[0073] Synthesis of substituted indigo-coumarin derivative IC-2d

[0074]

[0075] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced with N-(2-bromoethyl)-5-bromoindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-2d, yield: 48.9%.

[0076] 1 H NMR (400 MHz, CDC13) δ 7.64 (d, J = 9.2 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.14 (d, J = 9.3 Hz, 2H), 6.82 (t, J = 8.0 Hz, 2H), 6.77 (s, 1H), 6.25 (d, J = 9.3 Hz, 1H), 4.01 (s, 2H), 3.81 (s, 3H), 3.75 (d, J = 7.2 Hz, 2H), 1.88 (s, 2H), 1.83 - 1.76 (m, 2H), 1.60 (d, J = 6.5 Hz, 2H). 13C NMR (101 MHz, CDC13) δ 181.75, 160.91, 160.82, 157.74, 155.66, 149.92, 143.24, 140.56, 129.06, 128.19, 118.72, 116.85, 113.77, 113.23, 112.71, 112.36, 101.59, 66.24, 40.02. ESI-MS m / z: 415.3 [M+H] + .

[0077] Synthesis of substituted indigo-coumarin derivative IC-3d, Example 14

[0078]

[0079] The difference between Example 1 and this example is that N-(3-bromopropyl)-5- bromoindigo is used instead of N-(2-bromoethyl)-5-chloroindigo for the indigo intermediate of this example. Other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-3d, yield: 45.7%.

[0080] 1 H NMR (400 MHz, CDC13) δ 7.74 (s, 1H), 7.67 (t, J = 8.1 Hz, 2H), 7.40 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.80 (d, J = 8.1 Hz, 2H), 6.29 (d, J = 9.5 Hz, 1H), 4.12 (t, J = 5.6 Hz, 2H), 3.99 (t, J = 6.8 Hz, 2H), 2.30-2.25 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 182.07, 161.40, 161.08, 157.58, 155.78, 149.55, 143.32, 140.64, 129.00, 128.40, 118.70, 116.72, 113.53, 112.97, 112.49, 111.65, 101.52, 65.51, 37.54, 26.94. ESI-MS m / z: 429.2 [M+H] + .

[0081] Synthesis of substituted indigo-coumarin derivative IC-4d, Example 15

[0082]

[0083] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced by N-(5-bromopentyl)-5-bromoindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations refer to Example 1 to obtain the substituted indigo-coumarin derivative IC-5d, yield: 38.6%.

[0084] 1 H NMR (500 MHz, CDC13) δ 7.73-7.70 (m, 2H), 7.64 (d, J = 9.5 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 6.86-6.83 (m, 1H), 6.81 (dd, J = 8.6, 2.4 Hz, 1H), 6.78 (d, J = 2.3 Hz, 1H), 6.26 (d, J = 9.5 Hz, 1H), 4.07 (t, J = 5.4 Hz, 2H), 3.82 (t, J = 6.6 Hz, 2H), 1.93 (dd, J = 7.8, 5.4 Hz, 4H). 13 C NMR (101 MHz, CDC13) δ 182.18, 161.89, 161.16, 157.54, 155.86, 149.45, 143.38, 140.60, 128.87, 128.37, 118.79, 116.67, 113.26, 112.71, 112.51, 111.77, 101.44, 67.53, 39.97, 26.28, 23.93. ESI-MS m / z: 443.3 [M+H] + .

[0085] Example 16 Synthesis of substituted indigo-coumarin derivative IC-5d

[0086]

[0087] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced by N-(5-bromopentyl)-5-bromoindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations refer to Example 1 to obtain the substituted indigo-coumarin derivative IC-5d, yield: 38.6%.

[0088] 1H NMR (400 MHz, DMSO-d6) δ 7.77 (dd, J = 15.6, 8.1 Hz, 2H), 7.69 - 7.63 (m, 2H), 7.39 (d, J = 8.3 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 5.87 (s, 1H), 4.20 (t, J = 5.9 Hz, 2H), 3.71 (t, J = 6.7 Hz, 2H), 1.90 - 1.82 (m, 2H), 1.69 (dd, J = 14.2, 7.0 Hz, 2H), 1.54 (dd, J = 14.4, 7.3 Hz, 2H). 13 C NMR (126 MHz, DMSO) δ 182.71, 165.38, 162.10, 158.20, 153.19, 150.05, 140.19, 133.16, 127.10, 124.61, 123.24, 119.71, 116.90, 115.68, 115.28, 113.37, 90.93, 69.71, 27.99, 26.69, 23.04. ESI-MS m / z: 457.3 [M+H] + .

[0089] Synthesis of substituted indigo-coumarin derivative IC-3e

[0090]

[0091] The difference between this example and Example 1 is that N-(3-bromopropyl)-6-chloroindigo is used instead of N-(2-bromoethyl)-5-chloroindigo for the indigo intermediate of this example, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-3e, yield: 43.9%.

[0092] 1 H NMR (400 MHz, DMSO-d6) δ 7.99 (d, J = 9.5 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 1.6 Hz, 1H), 7.13 (dd, J = 8.0, 1.7 Hz, 1H), 6.94 - 6.86 (m, 2H), 6.29 (d, J = 9.5 Hz, 1H), 4.16 (t, J = 6.0 Hz, 2H), 3.86 (t, J = 6.6 Hz, 2H), 2.09 (p, J = 6.3 Hz, 2H). 13CNMR(101MHz,DMSO-d6)δ182.52,161.99,160.74,158.91,155.81,152.34,144.81,142.86,129.9 7,126.26,123.32,116.99,113.13,112.96,112.86,111.53,101.67,66.16,37.21,26.73.ESI-MS m / z:384.8[M+H] + .

[0093] Example 18 Synthesis of Substituted Isatin-Coumarin Derivative IC-4e

[0094]

[0095] The difference from Example 1 is that the indigo intermediate of this example is replaced by N-(4-bromobutyl)-6-chloroinsatin instead of N-(2-bromoethyl)-5-chloroinsatin. Other parameters and operations are the same as those in Example 1. The substituted indigo-coumarin derivative IC-4e is obtained with a yield of 51.7%.

[0096] 1 H NMR (400MHz, DMSO-d6) δ7.98(d,J=9.5Hz,1H),7.64-7.58(m,1H),7.55(d,J=8.0Hz,1H),7.42(d,J=1.6Hz,1H),7.16(dd,J=8.0,1.7Hz,1H) ,6.98(d,J=2.3Hz,1H),6.93(dd,J=8.6,2.4Hz,1H),6.28(d,J=9.5Hz,1H),4.12(t,J=6.2Hz,2H),3.75(t,J=6.8Hz,2H),1.89-1.66(m,4H). 13 C NMR(101MHz,DMSO-d6)δ182.54,162.22,160.76,158.82,155.87,152.35,144.79,142.91,129.9 4,126.29,123.36,116.92,113.17,112.88,112.76,111.56,101.62,68.37,26.14,23.86.ESI-MS m / z:398.8[M+H] + .

[0097] Example 19 Synthesis of Substituted Isatin-Coumarin Derivative IC-5e

[0098]

[0099] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced by N-(5-bromopentyl)-6-chloroindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-5e, yield: 47.3%.

[0100] 1 H NMR (400 MHz, CDC13) δ 7.63 (d, J = 9.5 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.91 (s, 1H), 6.85-6.72 (m, 2H), 6.24 (d, J = 9.5 Hz, 1H), 4.03 (t, J = 6.1 Hz, 2H), 3.76 (t, J = 7.2 Hz, 2H), 1.94-1.85 (m, 2H), 1.80 (dt, J = 14.9, 7.3 Hz, 2H), 1.70-1.49 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 181.91, 162.14, 161.21, 158.18, 155.89, 151.87, 144.82, 143.41, 128.79, 126.56, 123.94, 115.92, 113.09, 112.87, 112.55, 110.87, 101.37, 68.08, 40.25, 28.49, 26.92, 23.41. ESI-MS m / z: 412.8 [M+H] + .

[0101] Synthesis of substituted indigo-coumarin derivative IC-3f

[0102]

[0103] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced by N-(5-bromopentyl)-6-chloroindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-5e, yield: 47.3%.

[0104] 1H NMR (400 MHz, CDC13) δ 7.71 (d, J = 1.5 Hz, 1H), 7.66 (dd, J = 6.7, 1.5 Hz, 1H), 7.51 (d, J = 7.0 Hz, 1H), 6.89 (d, J = 6.7 Hz, 1H), 6.81 (dd, J = 7.0, 1.9 Hz, 1H), 6.77 (d, J = 1.9 Hz, 1H), 6.15 (s, 1H), 4.11 (t, J = 4.4 Hz, 2H), 3.98 (t, J = 5.4 Hz, 2H), 2.41 (s, 3H), 2.28 - 2.23 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 182.12, 161.24, 157.65, 155.13, 152.60, 149.56, 140.65, 133.78, 128.32, 125.80, 118.72, 116.67, 113.98, 112.21, 111.73, 109.81, 101.46, 65.47, 37.56, 26.92, 18.75. ESI-MS m / z: 443.2 [M+H] + .

[0105] Synthesis of substituted indigo-coumarin derivative IC-4f

[0106]

[0107] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced by N-(4-bromobutyl)-5-bromoindigo instead of N-(2-bromoethyl)-5-chloroindigo, the coumarin derivative is replaced by 4-methyl-7-hydroxycoumarin instead of 7-hydroxycoumarin, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-4f, yield: 48.1%.

[0108] 1 H NMR (400 MHz, CDC13) δ 7.71 (d, J = 1.5 Hz, 1H), 7.66 (dd, J = 6.7, 1.5 Hz, 1H), 7.51 (d, J = 7.0 Hz, 1H), 6.89 (d, J = 6.7 Hz, 1H), 6.81 (dd, J = 7.0, 1.9 Hz, 1H), 6.77 (d, J = 1.9 Hz, 1H), 6.15 (s, 1H), 4.11 (t, J = 4.4 Hz, 2H), 3.98 (t, J = 5.4 Hz, 2H), 2.41 (s, 3H), 2.28 - 2.23 (m, 2H). 13C NMR (101 MHz, CDC13) δ 182.23, 161.68, 161.29, 157.52, 155.18, 152.62, 149.44, 140.63, 128.31, 125.67, 118.73, 116.65, 113.70, 112.40, 112.03, 111.84, 101.39, 67.47, 39.98, 26.28, 23.93, 18.74. ESI-MS m / z: 457.3 [M+H] + .

[0109] Synthesis of substituted indigo-coumarin derivative IC-5f

[0110]

[0111] The difference between this example and Example 1 is that N-(5-bromopentyl)-5- bromoindigo is used instead of N-(2-bromoethyl)-5-chloroindigo for the indigo intermediate, 4-methyl-7-hydroxycoumarin is used instead of 7-hydroxycoumarin for the coumarin derivative, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-5f, yield: 39.7%.

[0112] 1 H NMR (400 MHz, CDC13) δ 7.72-7.69 (m, 2H), 7.50 (d, J = 7.0 Hz, 1H), 6.87-6.82 (m, 2H), 6.77 (d, J = 2.8 Hz, 1H), 6.15 (s, 1H), 4.02 (t, J = 4.9 Hz, 2H), 3.78 (t, J = 5.7 Hz, 2H), 2.42 (s, 3H), 1.93-1.86 (m, 2H), 1.84-1.76 (m, 2H), 1.62-1.56 (m, 2H). 13 C NMR (400 MHz, CDC13) δ 182.35, 161.90, 161.37, 157.49, 155.21, 152.67, 149.54, 140.59, 128.31, 125.60, 118.73, 116.57, 113.55, 112.57, 111.92, 111.83, 101.26, 67.95, 40.18, 29.74, 28.52, 26.89, 23.40. ESI-MS m / z: 471.3 [M+H] + .

[0113] Synthesis of substituted indigo-coumarin derivative IC-3g

[0114]

[0115] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced with N-(3-bromopropyl)-5-methoxyindigo instead of N-(2-bromoethyl)-5-chloroindigo, the coumarin derivative is replaced with 4-methyl-7-hydroxycoumarin instead of 7-hydroxycoumarin, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-3g, yield: 53.4%.

[0116] 1 H NMR (400 MHz, DMSO-d6) δ 7.64 (d, J = 6.9 Hz, 1H), 7.23 (dt, J = 7.2, 3.6 Hz, 1H), 7.15 (d, J = 6.9 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 6.95 - 6.89 (m, 2H), 6.19 (s, 1H), 4.10 (t, J = 4.8 Hz, 2H), 3.77 (s, 3H), 3.71 (t, J = 5.2 Hz, 2H), 2.38 (s, 3H), 1.79 (ddd, J = 11.0, 9.3, 5.5 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 184.15, 162.04, 160.60, 158.61, 156.09, 155.17, 153.84, 144.87, 126.85, 124.24, 118.44, 113.48, 112.85, 112.21, 111.53, 109.65, 101.59, 68.26, 56.32, 26.21, 23.87, 18.58. ESI-MS m / z: 394.4 [M+H] + .

[0117] Example 24 Synthesis of substituted indigo-coumarin derivative IC-4g

[0118]

[0119] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced with N-(3-bromopropyl)-5-methoxyindigo instead of N-(2-bromoethyl)-5-chloroindigo, the coumarin derivative is replaced with 4-methyl-7-hydroxycoumarin instead of 7-hydroxycoumarin, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-3g, yield: 53.4%.

[0120] 1H NMR (400MHz, DMSO-d6) δ7.66(d,J=7.0Hz,1H),7.24(dd,J=6.9,2.1Hz,1H),7.17(d,J=6.9Hz,1H),7.12(d,J=2.1Hz,1H),6.98-6.91(m, 2H), 6.21 (d, J = 0.7Hz, 1H), 4.12 (t, J = 4.9Hz, 2H), 3.77 (s, 3H), 3.72 (t, J = 5.3Hz, 2H), 2.39 (s, 3H), 1.80 (ddd, J = 11.7, 6.7, 4.0Hz, 4H). 13 C NMR (101MHz, DMSO-d6) δ184.24,162.07,160.62,158.63,156.10,155.19,153.90,144.89,126.91,124. 25,118.47,113.50,112.89,112.24,111.54,109.67,101.63,68.27,56.34,26.20,23.86,18.60.ESI-MS m / z:408.4[M+H] + .

[0121] Example 25 Synthesis of Substituted Isatin-Coumarin Derivative IC-5g

[0122]

[0123] The difference from Example 1 is that the indigo intermediate of this example is replaced by N-(5-bromopentyl)-5-methoxyisatin instead of N-(2-bromoethyl)-5-chloroisatin, and the coumarin derivative is replaced by 4-methyl-7-hydroxycoumarin instead of 7-hydroxycoumarin. Other parameters and operations are the same as those of Example 1. A substituted indigo-coumarin derivative IC-5 g is obtained with a yield of 49.4%.

[0124] 1 H NMR(400MHz,DMSO-d6)δ7.64(s,1H),7.31-7.06(m,3H),6.92(s,2H),6.19(s,1H),4. 05(s,2H),3.71(d,J=37.6Hz,5H),2.38(s,3H),1.72(d,J=40.0Hz,4H),1.48(s,2H). 13C NMR (101 MHz, DMSO-d6) δ 184.23, 162.15, 160.61, 158.52, 156.09, 155.18, 153.85, 144.90, 126.85, 124.31, 118.36, 113.44, 112.82, 112.27, 111.51, 109.68, 101.55, 68.53, 56.32, 28.52, 26.89, 23.16, 18.59. ESI-MS m / z: 422.4 [M+H] + .

[0125] Example 26 Synthesis of Substituted Indigo-Coumarin Derivative IC-3h

[0126]

[0127] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced by N-(3-bromopropyl)-indigo instead of N-(2-bromoethyl)-5-chloroindigo, the coumarin derivative is replaced by 4-methyl-7-hydroxycoumarin instead of 7-hydroxycoumarin, and other parameters and operations refer to Example 1 to obtain substituted indigo-coumarin derivative IC-3h, yield: 45.2%.

[0128] 1 H NMR (400 MHz, DMSO-d6) δ 7.63 (dd, J = 12.6, 6.6 Hz, 2H), 7.54 (d, J = 5.6 Hz, 1H), 7.21 (d, J = 6.1 Hz, 1H), 7.11 (t, J = 5.7 Hz, 1H), 6.89 (s, 2H), 6.19 (s, 1H), 4.16 (s, 2H), 3.86 (s, 2H), 2.38 (s, 3H), 2.11 (d, J = 4.2 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 184.23, 162.15, 160.61, 158.52, 156.09, 155.18, 153.85, 144.90, 126.85, 124.31, 118.36, 113.44, 112.82, 112.27, 111.51, 109.68, 101.55, 68.53, 56.32, 28.52, 26.89, 23.16, 18.59. ESI-MS m / z: 422.4 [M+H] + .

[0129] Example 27 Synthesis of Substituted Indigo-Coumarin Derivative IC-4h

[0130]

[0131] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced with N-(4-bromobutyl)-indigo instead of N-(2-bromoethyl)-5-chloroindigo, the coumarin derivative is replaced with 4-methyl-7-hydroxycoumarin instead of 7-hydroxycoumarin, and other parameters and operations refer to Example 1 to obtain a substituted indigo-coumarin derivative IC-4h, yield: 47.5%.

[0132] 1 H NMR (400 MHz, DMSO-d6) δ 7.65 (ddd, J = 7.0, 5.5, 2.2 Hz, 2H), 7.53 (d, J = 5.7 Hz, 1H), 7.22 (d, J = 6.4 Hz, 1H), 7.12 (t, J = 6.0 Hz, 1H), 6.93 (dd, J = 10.2, 3.2 Hz, 2H), 6.19 (s, 1H), 4.11 (t, J = 4.8 Hz, 2H), 3.74 (t, J = 5.3 Hz, 2H), 2.38 (s, 3H), 1.87 - 1.74 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 183.89, 162.06, 160.60, 158.62, 155.17, 153.85, 151.11, 138.55, 126.86, 124.88, 123.54, 117.98, 113.48, 112.86, 111.53, 111.14, 101.59, 68.29, 26.23, 23.90, 18.59. ESI-MS m / z: 378.4 [M+H] + .

[0133] Synthesis of substituted indigo-coumarin derivative IC-5h

[0134]

[0135] The difference between this example and Example 1 is that the indigo intermediate of this example is replaced with N-(4-bromobutyl)-indigo instead of N-(2-bromoethyl)-5-chloroindigo, the coumarin derivative is replaced with 4-methyl-7-hydroxycoumarin instead of 7-hydroxycoumarin, and other parameters and operations refer to Example 1 to obtain a substituted indigo-coumarin derivative IC-4h, yield: 47.5%.

[0136] 1 H NMR (400 MHz, DMSO-d6) δ 7.65 (ddd, J = 7.0, 5.5, 2.2 Hz, 2H), 7.53 (d, J = 5.7 Hz, 1H), 7.22 (d, J = 6.4 Hz, 1H), 7.12 (t, J = 6.0 Hz, 1H), 6.93 (dd, J = 10.2, 3.2 Hz, 2H), 6.19 (s, 1H), 4.11 (t, J = 4.8 Hz, 2H), 3.74 (t, J = 5.3 Hz, 2H), 2.38 (s, 3H), 1.87 - 1.74 (m, 4H).13 C NMR (101 MHz, DMSO-d6) δ 183.96, 162.16, 160.62, 158.54, 155.19, 153.86, 151.15, 138.64, 126.86, 124.91, 123.56, 117.90, 113.44, 112.83, 111.51, 111.20, 101.55, 68.57, 28.54, 26.94, 23.21, 18.59. ESI-MS m / z: 392.4 [M+H] + .

[0137] Inhibitory effect of substituted indigo-coumarin derivatives on monoamine oxidase-B

[0138] The inhibitory activity of the substituted indigo-coumarin derivatives obtained in Examples 1-28 on monoamine oxidase-B (MAO-B) was determined by fluorescence spectrophotometry. The experimental results were expressed as inhibition rate, with ladostigil as a positive control. All tests were performed on a PowerWave XS2 full-wavelength microplate reader, and the absorbance value was determined at 490 nm. The test concentration of the compounds was 40 μM, and the inhibition rate was calculated according to the following formula: inhibition rate (%) = [1- (sample-sample background) / (blank group-blank background)] x 100%. Among them, the blank group used 10 μL of PBS (pH = 7.6) instead of 10 μL of sample solution, the blank background group used 30 μL of PBS (pH = 7.6) instead of 30 μL of substrate, 10 μL of PBS (pH = 7.6) instead of 10 μL of sample solution, and 30 μL of PBS (pH = 7.6) instead of 30 μL of substrate.

[0139] (1) Preparation of sample solution:

[0140] Respectively, the sample was dissolved in dimethyl sulfoxide (DMSO, dimethyl sulfoxide) to prepare a 10 mM concentration, and stored in a low-temperature refrigerator at -20°C. Before use, it was diluted to the required concentration with phosphate buffer (0.2 mol / L, pH 7.6) to make the final concentration of DMSO less than or equal to 0.5% (v / v).

[0141] (2) Preparation of enzyme stock solution:

[0142] Monoamine oxidase B was purchased from Sigma Company; a certain amount of monoamine oxidase B was weighed and diluted with deionized water to the appropriate activity range.

[0143] (3) Preparation of substrate stock solution:

[0144] Tyramine was purchased from Sigma. A certain amount of tyramine was weighed and prepared into a 2.5 mM solution with phosphate buffer solution (0.2 mol / L, pH 7.6), and stored at 4° C. in the dark.

[0145] (4) Preparation of color developer stock solution:

[0146] A certain amount of vanillic acid, 4-aminoantipyrine and horseradish peroxidase were weighed and prepared into a color developing solution (1 mM vanillic acid, 0.5 mM 4-aminoantipyrine and 4 U / mL horseradish peroxidase) with phosphate buffer solution (0.2 mol / L, pH 7.6), and stored at 4°C in the dark.

[0147] (5) Testing:

[0148] In a 96-well plate, 10 μL of enzyme solution and 10 μL of sample solution were added, respectively, and incubated at 37°C for 20 min. Immediately, 30 μL of substrate and 10 μL of color development solution were added. After incubation at 37°C for 60 min, the absorbance value was measured at λ = 490 nm using a microplate reader. The experimental results are shown in Table 1.

[0149] Table 1 Inhibitory activity of substituted isatin-coumarin derivatives against MAO-B

[0150] Compound Inhibition rate (%) Compound Inhibition rate (%) IC-2a 27.2±1.5 IC-5d 29.3±0.7 IC-3a 31.2±1.3 IC-3e 43.9±1.3 IC-4a 36.5±1.1 IC-4e 47.5±1.2 IC-5a 28.6±0.9 IC-5e 42.6±1.1 IC-2b 54.3±1.4 IC-3f 49.8±1.2 IC-3b 62.9±0.8 IC-4f 52.3±1.0 IC-4b 68.4±0.6 IC-5f 42.6±0.9 IC-5b 58.8±1.2 IC-3g 84.1±1.1 IC-2c 67.3±1.3 IC-4g 89.3±0.8 IC-3c 78.6±0.8 IC-5g 73.4±0.9 IC-4c 82.5±0.9 IC-3h 68.7±1.0 IC-5c 71.2±1.1 IC-4h 71.8±0.7 IC-2d 28.1±1.2 IC-5h 63.1±1.2 IC-3d 32.7±1.0 7-hydroxycoumarin 45.1±1.1 IC-4d 40.1±1.4 Ladostigil 56.2±0.8

[0151] As shown in the table, all compounds obtained in this invention exhibit varying degrees of MAO-B inhibition, with compound IC-4g exhibiting the strongest inhibitory activity, reaching 89.3%, exceeding the positive control, ladostinigil. This demonstrates that the substituted isatin-coumarin derivatives obtained in this invention can be used to prepare anti-Alzheimer's disease drugs based on their MAO-B inhibition.

[0152] In addition, according to the compound structure and the overall structure-activity relationship in Table 1, the following may be true: the activity of the isatin part with electron-donating group substitution is higher than that of the electron-withdrawing group substitution; the activity of the coumarin part with methoxy group substitution is better; the chain length between the isatin part and the coumarin part has a greater influence on the activity, and the activity is better when the carbon chain length is 4.

[0153] Experimental Example 2: Inhibition of Aβ by Substituted Isatin-Coumarin Derivatives 1-42 Self-aggregation

[0154] The inhibitory effect of the substituted isatin-coumarin derivatives obtained in Examples 1 to 28 on Aβ was determined by ThT method. 1-42 Self-aggregation activity, compounds and Aβ 1-42 The final concentration of each was 20 μM. Curcumin and 7-hydroxycoumarin were used as positive controls. The specific steps included:

[0155] 1. Preparation of solution:

[0156] (1) 20mM pH 7.4 phosphate buffer solution (PBS): weigh 3.618g Na2HPO4 and 0.6027g KH2PO4, add 100mL ultrapure water, after the solid is completely dissolved, use ultrapure water to constant volume to 200mL, adjust the pH value of the solution to 7.4.

[0157] (2) Aβ 1-42 Protein solution: 1mg of protein is dissolved in 100μL of 1% NH4OH solution, the solution concentration is 2300μM, and it is stored in a-80℃ refrigerator for standby. When used, it is diluted to 40μM with PBS buffer.

[0158] (3) 50mM glycine-NaOH buffer solution: weigh 0.938g glycine, dissolve in 250mL of ultrapure water, and adjust the pH to 8.50 with 1mol / L NaOH solution, and store in a 4℃ refrigerator.

[0159] (4) 5μM thioflavin T solution (freshly prepared): weigh 2.2mg thioflavin T powder and dissolve in 689μL of pH 8.5 glycine-NaOH buffer solution, ultrasonic to completely dissolve the solid, and avoid light.

[0160] (5) Preparation of compound solution: accurately weigh a proper amount of compound with a precision analytical balance, dilute with DMSO to a transparent solution with a concentration of 10mM, and dilute to the test concentration with phosphate buffer when used.

[0161] 2. Inhibition activity test:

[0162] Take 10μL of 40μM Aβ 1-42 protein and 10μL of 40μM compound with a concentration of 40μM, place in an incubator, and incubate at 37℃ for 48h. The blank control is 10μL of 40μM Aβ 1-42 protein mixed with 10μL of pH 7.4 phosphate buffer solution for incubation together; the positive control is Aβ 1-42 protein and resveratrol for incubation together. After 72h, transfer the incubation solution to a black 96-well plate, add 180μL of 5μM thioflavin T solution, and stand in the dark at room temperature for 5min of reaction. Finally, measure the fluorescence absorption value with a multifunctional enzyme marker, wherein the excitation wavelength is 450nm, and the absorption wavelength is 485nm. The fluorescence intensity of Aβ 1-42 combined with thioflavin T in the negative control test is taken as the control, and the inhibition rate of the compound on Aβ 1-42 protein aggregation is calculated. The results are shown in Table 2. The results show that the compound of the present application has an inhibition rate of 40% on Aβ 1-42The compounds have strong inhibitory effect, and are better than 7-hydroxycoumarin and curcumin in activity. Among them, IC-4g and IC-3g exhibit the strongest inhibitory activity, reaching 88.9% and 83.4% respectively. It is proved that the substituted isatin-coumarin derivatives have a development prospect, and can be used for preparing drugs for resisting Alzheimer's disease based on inhibiting the self-aggregation of Aβ 1-42 self-aggregation, and are used for preparing drugs for resisting Alzheimer's disease.

[0163] In addition, according to the structure of the compound and the general structure-activity relationship in Table 2, it can be concluded that the activity of the compound is higher when the aromatic ring of the isatin and coumarin part is substituted with an electron-donating group than when it is substituted with an electron-withdrawing group; the length of the chain between the isatin part and the coumarin has an effect on the activity, and the activity is better when the carbon chain length is 4.

[0164] Table 2 Inhibitory activity of substituted isatin-coumarin derivatives on Aβ 1-42 self-aggregation

[0165] Compound Aβ 1-42 Self-aggregation inhibition rate Compound Aβ 1-42 Self-aggregation inhibition rate IC-2a 45.4±1.1 IC-5d 50.8±0.3 IC-3a 47.3±1.0 IC-3e 50.1±0.6 IC-4a 49.2±1.1 IC-4e 54.9±1.0 IC-5a 46.1±0.7 IC-5e 48.2±0.5 IC-2b 53.0±0.9 IC-3f 55.1±0.4 IC-3b 64.6±1.1 IC-4f 62.8±1.0 IC-4b 69.5±0.8 IC-5f 52.5±0.3 IC-5b 63.2±1.0 IC-3g 83.4±0.5 IC-2c 63.2±1.2 IC-4g 88.9±1.0 IC-3c 76.4±1.0 IC-5g 78.8±0.8 IC-4c 82.6±0.9 IC-3h 70.2±1.0 IC-5c 71.3±0.6 IC-4h 73.7±0.5 IC-2d 46.5±1.1 IC-5h 65.8±1.1 IC-3d 51.4±0.7 Curcumin 45.3±0.9 IC-4d 58.6±1.2 7-hydroxycoumarin 40.7±0.6

[0166] Experimental Example 3 In vitro antioxidant activity experiment of substituted isatin-coumarin derivatives

[0167] The in vitro antioxidant activity of the substituted isatin-coumarin derivatives obtained in Examples 1 to 28 is determined by using the ORAC method, AAPH is used as the source of peroxy free radicals, and sodium fluorescein (FL) is used as the fluorescence indicator to evaluate the antioxidant capacity of the partial compounds, and the experimental results are expressed in the equivalent of Trolox. The specific steps include the following:

[0168] (1) Preparation of phosphate buffer (PBS): an appropriate amount of phosphoric acid is weighed and diluted with ultrapure water to obtain a 75mM phosphoric acid solution; 8.56g of potassium phosphate dibasic is weighed and dissolved in 500mL of ultrapure water, and the pH is adjusted to 7.4 with the phosphoric acid solution to obtain a 75mM phosphate buffer with pH 7.4.

[0169] (2) AAPH solution (freshly prepared): 0.0588g of AAPH is accurately weighed, dissolved and diluted with 5.42mL of phosphate buffer, and a 40.0mM AAPH solution is prepared.

[0170] (3) Preparation of sodium fluorescein solution: 0.0650g of sodium fluorescein (FL) is accurately weighed and dissolved in 50mL of high-purity water to prepare a 3.4mM FL solution, which is stored in a 4°C refrigerator. When used, 2μL of the above solution is taken and dissolved in 50mL of phosphate buffer solution to obtain a 136nM FL solution.

[0171] (4) Preparation of Trolox solution: precisely weigh 2.50 mg of Trolox, and dissolve it in 1000 μL of DMSO by using a pipette to obtain a 10 mM Trolox solution; during the experiment, the Trolox DMSO solution is precisely pipetted and diluted with phosphate buffer to the test concentration.

[0172] (5) Preparation of compound solution: accurately weigh a proper amount of compound by using a precision analytical balance, dilute it with DMSO to a transparent solution with a concentration of 1 mM, and dilute it with phosphate buffer to the concentration used during use.

[0173] (6) Antioxidant activity test: 20 μL of compound or Trolox at different concentrations and 120 μL of FL diluent are pipetted into a black 96-well culture plate, mixed by using a pipette, incubated at 37°C for 15 min, and then 60 μL of AAPH is quickly added, and the fluorescence value is measured and recorded every 1 min by using a multifunctional enzyme marker, the excitation wavelength is 485 nm, and the emission wavelength is 535 nm, and the recording is performed for a total of 240 min. The blank control is replaced by 20 μL of PBS instead of the compound test. The area (AUC) between the curve and the coordinate is calculated by integration by using ORIGIN software, the protection area of the sample is calculated according to the formula: NetAUC = AUC antioxidant - AUC blank, the ORAC-FL value is calculated: [(AUC Sample-AUC blank) / (AUC Trolox-AUC blank)] / [concentration of Trolox / concentration of sample)], and the ORAC value of the sample is expressed in terms of Trolox value equivalent.

[0174] Table 3 in vitro antioxidant activity of substituted indigo-coumarin derivatives

[0175]

[0176] As shown in Table 3, some of the compounds obtained in the present application have good antioxidant effect in vitro, among which the ORAC value of compounds IC-5g and IC-4g is even as high as 3 or more at a concentration of 5 μM, and the antioxidant property is good. It is proved that the substituted indigo-coumarin derivatives obtained in the present application can be used for preparing drugs for resisting Alzheimer's disease based on the antioxidant effect.

[0177] Experimental Example 4 Inhibition of substituted indigo-coumarin derivatives on acetylcholinesterase

[0178] The inhibition of substituted indigo-coumarin derivatives obtained in Examples 1-28 on acetylcholinesterase is determined by using the Ellman method, and the results are expressed by IC 50Values are expressed as a percentage of the control, with tacrine as the positive control. All tests were performed in a PowerWave XS2 microplate reader, measuring at 37°C. Data analysis was performed using the software Origin. The procedure was as follows:

[0179] (1) Preparation of drug solution:

[0180] A certain amount of each sample to be analyzed was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM, and stored at -20°C. Before use, the sample was diluted with phosphate buffer (0.1 mol / L, pH 8.0) to the desired concentration, so that the final concentration of DMSO was less than or equal to 0.5% (v / v).

[0181] (2) Preparation of enzyme stock solution:

[0182] Acetylcholinesterase (E.C. 3.1.1.7, from electric ell.) was purchased from Sigma. A certain amount of acetylcholinesterase was diluted with deionized water to the appropriate activity range.

[0183] (3) Preparation of substrate stock solution:

[0184] Acetylthiocholine (ATC) was purchased from Sigma. A certain amount of ATC was dissolved in phosphate buffer (0.1 mol / L, pH 8.0) to a concentration of 0.01 mol / L, and stored at 4°C in the dark.

[0185] (4) Preparation of chromogenic agent stock solution:

[0186] Chromogenic agent 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) was purchased from Sigma. A certain amount of DTNB was dissolved in phosphate buffer (0.1 mol / L, pH 8.0) to a concentration of 0.01 mol / L, and stored at 4°C in the dark.

[0187] (5) Testing:

[0188] In 96-well plates, 6 wells were selected, 10 μL of enzyme solution was added, and 0, 5, 10, 20, 35, 50 μL of the compound solution to be tested was added, 0.1 mol / L pH 8.0 phosphate buffer solution was added to make the total volume 100 μL, and incubation was carried out at 37°C in a full-wavelength enzyme marker for 15 min, and then 10 μL of an ATP solution, 10 μL of a DTNB solution, and 80 μL of a phosphate buffer solution were added to make a total of 100 μL, and scanning was carried out at λ = 412 nm for 2 min to determine the absorbance change. The experimental results are shown in Table 4, and the results show that most of the compounds described in the application have good acetylcholinesterase inhibitory activity, and the activity is better than that of the mother nucleus 7-hydroxycoumarin. Among them, compounds IC-3a, IC-3d and IC-3e have the best acetylcholinesterase inhibitory activity, and the IC 50 values are all less than 10 μM, and can be used for preparing drugs for resisting Alzheimer's disease.

[0189] Table 4 Inhibitory activity of compounds on acetylcholinesterase

[0190] Compound IC 50 forAChE (μM) Compound IC 50 forAChE (μM) IC-2a 18.26±0.18 IC-5d 79.85±0.19 IC-3a 8.31±0.15 IC-3e 9.11±0.13 IC-4a 45.02±0.13 IC-4e 27.92±0.11 IC-5a >100 IC-5e 94.37±0.18 IC-2b 39.64±0.21 IC-3f 19.28±0.12 IC-3b 28.51±0.17 IC-4f 50.32±0.25 IC-4b 37.25±0.15 IC-5f >100 IC-5b >100 IC-3g 69.31±0.18 IC-2c 50.32±0.25 IC-4g 85.93±0.12 IC-3c 32.81±0.13 IC-5g n.d. a ]]> IC-4c 56.18±0.21 IC-3h 51.09±0.14 IC-5c >100 IC-4h 68.64±0.18 IC-2d 12.15±0.18 IC-5h n.d. a ]]> IC-3d 8.67±0.22 Tacrine 0.31±0.10 IC-4d 21.83±0.13 7-hydroxycoumarin 75.81±0.19

[0191] a Not determined

[0192] Metal complexing experiment of substituted indigo-coumarin derivative

[0193] The metal complexing ability of the substituted indigo-coumarin derivative IC-4g obtained in Example 24 was determined by UV-vis method, and the specific steps were as follows:

[0194] 1. Preparation of solution:

[0195] (1) Compound IC-4g solution: A certain amount of compound was weighed and prepared into 1 mM with anhydrous ethanol.

[0196] (2) Metal ion solution: A certain amount of NaCl, KCl, CaCl2, ZnSO4, CuSO4 and FeSO4 were weighed and prepared into 10 mM with ultrapure water, and then diluted to 1 mM with anhydrous ethanol.

[0197] 2. Action of compound IC-4g with NaCl, KCl, CaCl2, ZnSO4, CuSO4 and FeSO4

[0198] Take 6 5mL centrifuge tubes, respectively add 360μL 1mM IC-4g compound solution, then respectively add 360μL 1mM NaCl, KCl, CaCl2, ZnSO4, CuSO4 and FeSO4 solution, finally add anhydrous ethanol to make the total volume 3000μL, so that the final concentration of compound IC-4g and metal ions are both 120μM. Blank control is 360μL 1mM compound solution supplemented with anhydrous ethanol to the same concentration of compound in the sample. After mixing, place at room temperature for 30min, pour into quartz cuvettes and scan the absorption curve by ultraviolet-visible spectrometer. The test temperature is room temperature, the test range is 200-700nm, the wavelength interval is 1nm, the scanning rate is 200nm / min, each sample is tested three times and the average value is taken.

[0199] Results are shown in Figure 1 As shown in the figure, the compound IC-4g has strong metal complexing ability, and has good selectivity for Cu 2+ and Fe 2+ ; other substituted indigo-coumarin with similar structure also has similar effect, proving that the substituted indigo-coumarin derivative obtained in the application can be used for preparing anti-Alzheimer's drugs based on metal complexing effect.

[0200] Experimental Example 6: Toxicity of substituted indigo-coumarin derivative to nerve cells

[0201] The toxicity of substituted indigo-coumarin derivative obtained in Examples 1-28 to nerve cells (SH-SY5Y) is determined by MTT method, and the specific steps are as follows:

[0202] 1. Solution preparation

[0203] (1) DMEM culture medium: dissolve dry powder culture medium in 300mL ultrapure water in a 1000mL beaker, then rinse the inner surface of the package twice with 300mL ultrapure water, combine the solutions, and magnetically stir to completely dissolve; add 3.7g sodium bicarbonate and 2.38g HEPES, and magnetically stir to completely dissolve; adjust the pH to 7.5 with 10M sodium hydroxide under stirring, filter sterilize with a 0.22μM filter membrane in an ultraclean bench, and store in a 4℃ refrigerator; when used, add antibiotics (final concentration of penicillin is 100U / mL, and streptomycin is 100μg / mL) and fetal bovine serum (10%).

[0204] (2) PBS buffer solution: accurately weigh 8g NaCl, 0.2g KH2PO4 and 2.88g Na2HPO4·12H2O, dissolve and make up to 1L with ultrapure water, high-pressure sterilize at 120℃ for 20min, and store in a 4℃ refrigerator.

[0205] (3) MTT solution: MTT was prepared to 5 g / L with PBS solution, filtered with 0.22 μM filter membrane to remove bacteria, and stored in a 4℃ refrigerator in the dark.

[0206] 2. Culture of nerve cells SH-SY5Y

[0207] The nerve cell strain SH-SY5Y was taken, and DMEM medium was used to routinely culture the nerve cells in a culture box with a temperature of 37℃, saturated humidity, and an environment of 5% CO2 and 95% air, and the nerve cells were subcultured once every 2-3 days.

[0208] 3. Nerve cell toxicity determination

[0209] (1) Cells in the logarithmic growth phase were taken, digested with 0.25% trypsin, washed twice with PBS, resuspended with DMEM medium, counted under a microscope with a cell counting plate, and adjusted to a cell concentration of 5×10 4 6 / mL, inoculated in a 96-well cell culture plate at 100 μL / well, and cultured for 24 h to allow the cells to adhere.

[0210] (2) The original culture medium was removed, and compound solutions of different concentrations diluted with DMEM medium were added to each well at 100 μL, and 5 replicate wells were set. The blank and control groups were added with the medium instead of the compound, and placed in a 37℃, 5% CO2 incubator for culture for 48 h; 4 h before the end of the experiment, the medium containing 5 mg / mL MTT was added to the sample group and the control group at 100 μL / well, and the culture was continued for 4 h.

[0211] (3) The supernatant was discarded, DMSO was added to each well at 100 μL, and the product formazan was fully dissolved by oscillation, and the absorbance value (OD value) of each well was determined on a full-wavelength enzyme marker instrument at a wavelength of 570 nm; the cell survival rate (%) in each sample = (OD sample-OD blank) / (OD control-OD blank)×100%; the cell inhibition rate (%) of each sample = 100%-the cell survival rate (%) of each sample, and the inhibition rate was plotted against the concentration, and the concentration at which the inhibition rate was 50% was the IC 50 value of the compound. The results are shown in Table 5.

[0212] Table 5 Toxicity of substituted indigo-coumarin derivatives on nerve cells (SH-SY5Y)

[0213] Compound IC 50 (μM) Compound IC 50 (μM) IC-2a 124.3±0.6 IC-4d 130.9±0.4 IC-3a 128.1±0.8 IC-5d 128.7±0.5 IC-4a 109.8±0.3 IC-3e 142.3±0.6 IC-5a 106.4±0.5 IC-4e 137.5±0.4 IC-2b 101.5±0.4 IC-5e 131.2±0.6 IC-3b 105.3±0.6 IC-3f 163.4±0.5 IC-4b 114.5±0.3 IC-4f 171.3±0.4 IC-5b 109.6±0.5 IC-5f 149.1±0.3 IC-2c 141.3±0.5 IC-3g 228.3±0.4 IC-3c 136.5±0.8 IC-4g 217.7±0.5 IC-4c 139.2±0.4 IC-5g 210.9±0.3 IC-5c 121.6±0.8 IC-3h 132.5±0.8 IC-2d 134.8±0.5 IC-4h 143.7±0.2 IC-3d 133.2±0.6 IC-5h 136.2±0.4

[0214] As shown in Table 5, the IC 50 values of the substituted indigo-coumarin derivatives obtained in the application on SH-SY5Y cells are all greater than 100 μM, and the compounds exhibit low nerve cell toxicity; among them, the IC 50The value is 217.7μM, which has good safety.

[0215] Experimental Example 7: Effects of substituted isatin-coumarin derivatives IC-4c, IC-3g, and IC-4g on Aβ 1-42 Study on the neuroprotective effect of SH-SY5Y cell injury induced by

[0216] The MTT method was used to determine the effects of the substituted isatin-coumarin derivatives IC-4c, IC-3g and IC-4g obtained in Examples 11, 23 and 24 on Aβ 1-42 The neuroprotective effect of induced SH-SY5Y cell injury was investigated in the following steps:

[0217] SH-SY5Y cells were subcultured in 96-well plates with a cell density of 1 × 10 4 After removing the culture medium, Aβ 1-42 (20 μM) solution and 10 μM of different compounds (IC-4c, IC-3g and IC-4g) were incubated for 24 h, and curcumin was used as a positive control. Cell viability was determined by the MTT method of Experimental Example 5 above. The experimental results are shown in Figure 2 .

[0218] Depend on Figure 2 It can be seen that the substituted isatin-coumarin derivatives IC-4c, IC-3g and IC-4g obtained in the present invention have an inhibitory effect on Aβ 1-42 The compound IC-4g had the best neuroprotective effect against SH-SY5Y cell damage induced by Aβ. 1-42 After co-incubation with treated SH-SY5Y cells, the cell survival rate increased to 82.5%. Experimental Example 8 Effect of the substituted isatin-coumarin derivative IC-4g on Aβ 1-42 Study on the improvement of memory impairment induced by

[0219] The passive avoidance test was used to evaluate the effect of the substituted isatin-coumarin derivative IC-4g obtained in Example 24 on Aβ 1-42 The specific steps are as follows:

[0220] (1) Establishment of AD mouse model

[0221] Sixty male SD mice (3 months old) were placed in an environment with a temperature of 22-25°C, a relative humidity of 50-70%, and a 12-h light-dark cycle. The 60 male SD mice were randomly divided into 6 groups: a) control group (injected with normal saline and gavaged with distilled water); b) AD model group (Aβ 1-42, 410 pmol / 5 μL); c) positive control group (donepezil, 8.0 mg / kg); d) high-dose group (IC-4g-H 8.0 mg / kg); e) medium-dose group (IC-4g-M 4.0 mg / kg); f) low-dose group (IC-4g-L 2.0 mg / kg). Aβ 1-42 Forty-eight hours after injection, each treatment group received oral gavage once daily for 14 consecutive days. The control group and AD model group received distilled water via gavage using the same method. After 14 days of treatment, a passive avoidance test was performed using the following method.

[0222] (2) Passive avoidance experiment

[0223] The in vivo memory enhancement ability was assessed by a passive avoidance test in mice, which included two separate trials (a training trial and a test trial 24 hours later). The experimental apparatus consisted of two compartments of equal size (a light compartment and a dark compartment) separated by a guillotine door, and illuminated by a 250lx LED light in a light box. In the training trial, the mice were first placed in the light compartment and allowed to move freely for 5 minutes to familiarize themselves with the surrounding environment. The door was then opened, and the mice quickly entered the dark compartment. At the same time, the electric stimulator was activated, and the animals received an electric shock to their feet (24V, 0.5mA). The mice were trained repeatedly for 5 minutes. If the mice failed to enter the dark compartment within 180 seconds, they were eliminated from the test.

[0224] The test was conducted 24 hours after the training trial. We placed the mice back into the light compartment and opened the door. The total test time was set to 5 minutes. During the test time, the delay time and the number of errors were recorded. The delay time (i.e., latency) was the time it took the mouse to enter the dark compartment, and the number of errors was the number of times the mouse entered the dark compartment within 5 minutes. The results can be found in Figure 3 .

[0225] Depend on Figure 3 As shown, Aβ 1-42 The latency of the treatment group (model group, 95 sec) was significantly lower than that of the control group (189 sec), and the average number of errors within 5 minutes increased significantly (9.4 in the model group and 2.8 in the control group), indicating that the injection of Aβ 1-42 Significant memory impairment was observed in mice. Treatment with donepezil and different concentrations of compound IC-4g (2.0, 4.0, and 8.0 mg / kg) significantly reversed both latency and the average number of errors compared to the model group. Furthermore, compound IC-4g prolonged latency and reduced the number of errors in a dose-dependent manner. The 8.0 mg / kg dose of compound IC-4g demonstrated a superior effect in increasing latency and reducing the number of errors compared to the donepezil group.

[0226] As can be seen from the above, the obtained substituted indigo-coumarin derivative has good MAO-B inhibitory activity, inhibits Aβ self-aggregation, inhibits acetylcholinesterase, has antioxidant activity and metal chelation, and has less toxicity to nerve cells and high safety. Among them, the compound IC-4g has strong inhibitory activity on Aβ 1-42 induced SH-SY5Y cell damage, has strong neuroprotective effect; animal experiments show that high-dose compound IC-4g can effectively improve Aβ 1-42 induced memory impairment of mice. It can be seen that the compound provided by the present application is very suitable for preparing an anti-Alzheimer's disease drug.

[0227] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A novel substituted indigo-coumarin derivative, characterized in that, having the structure of formula (I): ， wherein R is selected from hydrogen, halogen, methoxy; R1is selected from hydrogen or methyl, and n is a positive integer from 1 to 4; and the compounds 5a and 5b are excluded from the structure of formula (I). The structural formula is: The structural formula of 5b is: ​ 2. Novel substituted indigo-coumarin derivatives according to claim 1, characterized in that wherein R is selected from hydrogen, 5-halogen, 5-methoxy, 6-halogen; R1is selected from hydrogen or methyl, and n is a positive integer from 1 to 4; and the compounds 5a and 5b are excluded from the structure.

3. The novel substituted indigo-coumarin derivative according to claim 1, wherein The substituted isatin-coumarin derivative has any one of the following structures: ， 。 4. A process for the preparation of novel substituted indigo-coumarin derivatives as claimed in any one of claims 1 to 3, characterized in that, The synthesis route is as follows: ， comprising the following steps: S1. Compound (II) and Br-(CH2) n -Br undergoes substitution reaction in an alkaline environment to obtain a compound of formula (III); S2, substituting the compound of formula (III) obtained in step S1 with substituted 7-hydroxycoumarin under alkaline conditions to obtain the compound of formula (I); wherein R, R1, and n are as defined in any one of claims 1-3.

5. Use of the novel substituted isatin-coumarin derivative according to any one of claims 1-3 in the preparation of a monoamine oxidase-B inhibitor.

6. Use of the novel substituted isatin-coumarin derivative according to any one of claims 1-3 in the preparation of an Aβ aggregation inhibitor.

7. Use of the novel substituted isatin-coumarin derivative according to any one of claims 1-3 in the preparation of an antioxidant.

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