Novel substituted isatin-coumarin derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a novel substituted isatin-coumarin derivative as well as a preparation method and application thereof. The novel substituted isatin-coumarin derivative has remarkable effects of inhibiting monoamine oxidase-B activity, inhibiting A beta aggregation, resisting oxidation, inhibiting acetylcholin esterase and chelating metal ions, and has a treatment effect on diseases related to the mechanisms; the compound disclosed by the invention has a remarkable treatment effect on a plurality of treatment targets of the Alzheimer's disease, particularly has a remarkable treatment effect on a plurality of treatment targets of the Alzheimer's disease, is relatively low in biotoxicity and high in safety, and has relatively high medical research and market application values. And on the other hand, production raw materials are cheap, reaction steps are few, and the preparation method is simple and is very suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a novel substituted isatin-coumarin derivative and a preparation method and application thereof. Background Art

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

[0003] Current studies have shown that the main pathological characteristics of AD patients are the aggregation of β-amyloid protein (Aβ) into senile plaques, abnormal aggregation of intracellular Tau protein to form neurofibrillary tangles (NFTs) and neuronal death. However, the cause of AD has not been clarified so far, involving many pathogenic factors; a variety of related hypotheses have been proposed in the process of research to try to explain the occurrence and development of the disease, including the cholinergic hypothesis, the amyloid protein cascade hypothesis, the Tau protein hypothesis, the oxidative stress hypothesis, the metal ion hypothesis, the neuroinflammation hypothesis, etc. And corresponding solutions have been proposed for these hypotheses. The first is the amyloid protein cascade hypothesis. Researchers have proposed a variety of treatment strategies for the metabolic process of Aβ. One of them is to design and synthesize small molecule ligands that can inhibit Aβ aggregation. The second is the cholinergic theory, which believes that the reduction of cholinergic nerve function is the main cause of AD. Based on this, scientists have developed cholinesterase inhibitors (ChEI). The third is the oxidative stress theory, which believes that abnormal material and energy metabolism in brain tissue leads to the accumulation of free radicals, oxidative damage, cell apoptosis and AD characteristic neuropathological changes. In response to this, researchers have proposed designing and synthesizing compounds with antioxidant capacity to solve the problem of oxidative stress. At present, this strategy has achieved good results. In addition, studies have shown that some metal ions such as copper, iron, and zinc 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 as high as that in normal people. In addition, metal ions such as copper and iron can promote the generation of ROS, leading to increased oxidative stress. Therefore, metal ion chelators have also 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 AD patients where the concentration is higher around senile plaques. The increased activity will lead to an increase in neurotoxic free radicals in the brain and oxidative stress, and at the same time further accelerate the aggregation of Aβ protein and excessive phosphorylation of tau protein in the brain, ultimately causing nerve damage and neuronal death. Therefore, MAO-B inhibitors have also been used to treat AD in recent years.

[0005] From the pathogenesis of AD, we can see that AD has multiple pathogenesis mechanisms, and each mechanism is closely related and influences each other. Therefore, the therapeutic effect of drugs targeting a single target is not ideal. However, more and more studies have shown that drugs that act on multiple targets related to the disease at the same time, namely multi-target drugs, may have better therapeutic effects. The design of multi-target drugs aims to use biological structure information and pharmacophore models to obtain the required multiple biological activities. The characteristic of multi-target drugs is that they act on different pathological and physiological links of the disease to enhance the efficacy. Therefore, designing and synthesizing multi-target drugs with anti-Aβ aggregation activity, metal ion complexation, inhibition of MAO-B activity, and antioxidant activity may be an effective strategy for the treatment of AD. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing single-target drugs for treating AD, and to provide a novel substituted isatin-coumarin derivative with multi-target therapeutic effects on AD, as well as a preparation method and application thereof.

[0007] The present invention provides a novel substituted isatin-coumarin derivative having a structure of formula (I):

[0008]

[0009] Wherein, 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 to 4.

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

[0011] Therefore, the present invention claims the use of the novel substituted isatin-coumarin derivatives in the preparation of monoamine oxidase-B inhibitors.

[0012] In addition, the present invention also claims to protect the use of the novel substituted isatin-coumarin derivatives in the preparation of acetylcholinesterase inhibitors.

[0013] In addition, the present invention also claims to protect the use of the novel substituted isatin-coumarin derivatives in the preparation of Aβ aggregation inhibitors.

[0014] In addition, the present invention also claims to protect the use of the novel substituted isatin-coumarin derivatives in the preparation of antioxidants.

[0015] In addition, the present invention claims the use of the novel substituted isatin-coumarin derivatives in the preparation of metal complexing agents.

[0016] Based on the therapeutic principles of inhibiting monoamine oxidase-B, inhibiting acetylcholinesterase, resisting Aβ aggregation, metal ion chelation and anti-oxidation, the novel substituted isatin-coumarin derivatives provided by the present invention have therapeutic effects on diseases related to the above mechanisms.

[0017] Therefore, the present invention also claims to protect the use of the novel substituted isatin-coumarin derivatives in the preparation of drugs for treating Alzheimer's disease, cerebrovascular dementia, myasthenia gravis, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis.

[0018] Furthermore, the dosage form of the pharmaceutical preparation is tablets, pills, capsules, injections, suspensions or emulsions.

[0019] Compared with the related art, the novel substituted isatin-coumarin derivatives provided by the present invention have the following beneficial effects:

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

[0021] Figure 1 This is the ultraviolet spectrum of the substituted isatin-coumarin derivative IC-4g interacting with metal ions in Experimental Example 24 of the present invention;

[0022] Figure 2 This is a graph showing the results of the MTT assay of the neuroprotective effect of the compound on Aβ-induced SH-SY5Y cell injury in Example 7;

[0023] Figure 3 This is a diagram of the passive avoidance experiment results in Example 8. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0025] Example 1 Synthesis of Substituted Isatin-Coumarin Derivative IC-2a

[0026]

[0027] 5 mL of DMF, 7-hydroxycoumarin (0.15 g, 0.93 mmol) and anhydrous potassium carbonate (0.24 g, 1.76 mmol) were added to a 25 mL round-bottom flask. After stirring and reacting at room temperature for 30 min, an indigo intermediate N-(2-bromoethyl)-5-chloroindigo (0.25 g, 0.88 mmol) was added. The reaction was continued to be stirred at 60° C. and the reaction progress was monitored by TLC. After the reaction, the reaction solution was poured into 40 mL of ice water, stirred at room temperature, and the pH of the solution was adjusted to about 7-8 with dilute hydrochloric acid. Stirring was continued for another 30 min to allow the solid to fully precipitate. The solid was then filtered and dried. The substituted indigo-coumarin derivative IC-2a was separated by silica gel column chromatography with a yield of 39.1%.

[0028] 1 H NMR (400MHz, CDCl3) δ7.64-7.55(m,3H),7.36(d,J=9.3Hz,1H),7.14(d,J=8.4Hz,1H),6. 75(d,J=6.7Hz,2H), 6.27(d,J=9.5Hz,1H), 4.32(t,J=4.9Hz,2H), 4.18(t,J=4.9Hz,2H). 13 C NMR (101MHz, CDCl3) δ182.13,161.24,161.08,157.95,155.68,149.31,143.12,137.87,129 .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] Example 2 Synthesis of Substituted Isatin-Coumarin Derivative IC-3a

[0030]

[0031] The difference from Example 1 is that the indigo intermediate of this example uses N-(3-bromopropyl)-5-chloroindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations are the same as those of Example 1 to obtain a substituted indigo-coumarin derivative IC-3a with a yield of 42.8%.

[0032] 1H NMR (400MHz, CDCl3) δ7.63(d,J=9.5Hz,1H),7.58(s,1H),7.51(d,J=8.4Hz,1H),7.38(d,J=8.2Hz,1H),6.90(d,J=8.3Hz,1H ), 6.78 (d, J = 7.9Hz, 2H), 6.27 (d, J = 9.5Hz, 1H), 4.11 (t, J = 5.5Hz, 2H), 3.97 (t, J = 6.8Hz, 2H), 2.24 (dd, J = 12.1, 6.1Hz, 2H). 13 CNMR (101MHz, CDCl3) δ182.20,161.41,161.00,157.82,155.83,149.10,143.26,137.76,129.74 ,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] Example 3 Synthesis of Substituted Isatin-Coumarin Derivative IC-4a

[0034]

[0035] The difference from Example 1 is that the indigo intermediate of this example uses N-(4-bromobutyl)-5-chloroindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations are the same as those of Example 1 to obtain a substituted indigo-coumarin derivative IC-4a with a yield of 52.1%.

[0036] 1 H NMR (400MHz, CDCl3) δ7.63(d,J=9.4Hz,1H),7.56(d,J=9.4Hz,2H),7.36(d,J=8.6Hz,1H),6.88(d,J=8.1Hz,1H) ,6.83-6.76(m,2H),6.26(d,J=9.3Hz,1H),4.08(d,J=5.3Hz,2H),3.82(d,J=6.5Hz,2H),1.92(d,J=3.1Hz,4H). 13C NMR (101MHz, CDCl3) δ182.34,161.89,161.13,157.71,155.86,149.00,143.36,137.74,129.70 ,128.87,125.51,118.46,113.27,112.70,111.34,101.45,67.53,39.98,26.28,23.94.ESI-MS m / z:398.7[M+H] + .

[0037] Example 4 Synthesis of Substituted Isatin-Coumarin Derivative IC-5a

[0038]

[0039] The difference from Example 1 is that the indigo intermediate of this example uses N-(5-bromopentyl)-5-chloroindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations are the same as those of Example 1 to obtain a substituted indigo-coumarin derivative IC-5a with a yield of 46.8%.

[0040] 1 H NMR (500MHz, CDCl3) δ7.63(d,J=9.5Hz,1H),7.59-7.52(m,2H),7.36(d,J=8.6Hz,1H),6.86(d,J=8.3Hz,1H),6.82-6.75(m,2H) ,6.25(d,J=9.5Hz,1H),4.01(t,J=6.2Hz,2H),3.77(t,J=7.2Hz,2H),1.91-1.85(m,2H),1.83-1.76(m,2H),1.62-1.53(m,2H). 13 C NMR (126MHz, CDCl3) δ182.41,162.11,160.95,157.71,155.89,149.11,143.42,137.70,129.62,128.7 9,125.50,118.45,113.12,112.88,112.55,111.33,101.32,68.02,40.19,28.50,26.89,23.39.ESI-MS m / z:412.5[M+H] + .

[0041] Example 5 Synthesis of Substituted Isatin-Coumarin Derivative IC-2b

[0042]

[0043] The difference from Example 1 is that the indigo intermediate of this example uses N-(2-bromoethyl)-isatin instead of N-(2-bromoethyl)-5-chloroisatin, and other parameters and operations are the same as those of Example 1 to obtain a substituted indigo-coumarin derivative IC-2b with a yield of 47.9%.

[0044] 1 HNMR(400MHz, CDCl3)δ7.67-7.61(m,3H),7.36(d,J=6.8Hz,1H),7.16(t,J=5.8Hz,2H), 6.80-6.72(m,2H),6.26(d,J=7.6Hz,1H),4.32(t,J=3.9Hz,2H),4.19(t,J=3.9Hz,2H). 13 C NMR (101MHz, CDCl3) δ182.87,160.99,160.91,158.49,155.69,151.12,143.21,138.35,129 .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] Example 6 Synthesis of Substituted Isatin-Coumarin Derivative IC-3b

[0046]

[0047] The difference from Example 1 is that the indigo intermediate of this example uses N-(3-bromopropyl)-insatin instead of N-(2-bromoethyl)-5-chloroinsatin, and other parameters and operations are the same as those of Example 1 to obtain a substituted indigo-coumarin derivative IC-3b with a yield of 50.8%.

[0048] 1 H NMR (400MHz, CDCl3) δ7.65-7.60(m,2H),7.54(t,J=7.8Hz,1H),7.37(d,J=8.5Hz,1H),7.11(t,J=7.5Hz,1H),6.94(d,J=7.9H z,1H),6.82-6.73(m,2H),6.26(d,J=9.5Hz,1H),4.11(t,J=5.7Hz,2H),3.97(t,J=6.7Hz,2H),2.27(dd,J=12.3,6.1Hz,2H). 13C NMR (101MHz, CDCl3): δ161.55,161.01,158.35,155.82,150.85,143.25,138.39,128.92,125.61 ,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] Example 7 Synthesis of Substituted Isatin-Coumarin Derivative IC-4b

[0050]

[0051] The difference from Example 1 is that the indigo intermediate of this example uses N-(4-bromobutyl)-isatin instead of N-(2-bromoethyl)-5-chloroisatin, and other parameters and operations are the same as those of Example 1 to obtain a substituted indigo-coumarin derivative IC-4b with a yield of 54.2%.

[0052] 1 H NMR (500MHz, CDCl3) δ7.65-7.58(m,3H),7.37(d,J=8.6Hz,1H),7.14(t,J=7.5Hz,1H),6.93(d,J=7.8Hz,1H) ,6.82(d,J=8.6Hz,1H),6.78(s,1H),6.26(d,J=9.5Hz,1H),4.08(s,2H),3.83(d,J=6.3Hz,2H),1.94(s,4H). 13 C NMR (126MHz, CDCl3) δ183.37,161.94,161.19,158.26,155.85,150.76,143.40,138.41,128.86,12 5.62,123.84,117.63,113.22,112.69,112.66,110.08,101.49,67.60,39.79,26.31,23.99.ESI-MS m / z:364.4[M+H] + .

[0053] Example 8 Synthesis of Substituted Isatin-Coumarin Derivative IC-5b

[0054]

[0055] The difference from Example 1 is that the indigo intermediate of this example uses N-(5-bromopentyl)-insatin instead of N-(2-bromoethyl)-5-chloroinsatin, and other parameters and operations are referred to Example 1 to obtain a substituted indigo-coumarin derivative IC-5b with a yield of 49.4%.

[0056] 1 H NMR (400MHz, CDCl3) δ7.66-7.57(m,3H),7.36(d,J=8.6Hz,1H),7.13(t,J=7.5Hz,1H),6.91(d,J=7.9Hz,1H),6.82(dd,J=8.6,2.3Hz,1H),6.77( d,J=2.3Hz,1H),6.25(d,J=9.5Hz,1H),4.02(t,J=6.2Hz,2H),3.78(t,J =13.5Hz,2H),1.92-1.86(m,2H),1.84-1.77(m,2H),1.63-1.55(m,2H). 13 C NMR (101MHz, CDCl3) δ183.49,162.15,161.27,158.22,155.88,150.88,143.45,138.37,128.79,125.5 9,123.76,117.62,113.08,112.88,112.52,110.08,101.35,68.10,40.02,28.54,26.99,23.43.ESI-MS m / z:378.4[M+H] + .

[0057] Example 9 Synthesis of Substituted Isatin-Coumarin Derivative IC-2c

[0058]

[0059] The difference from Example 1 is that the indigo intermediate of this example uses N-(2-bromoethyl)-5-methoxy indigo instead of N-(2-bromoethyl)-5-chloro indigo, and other parameters and operations are the same as those of Example 1 to obtain a substituted indigo-coumarin derivative IC-2c with a yield of 49.6%.

[0060] 1H NMR (400MHz, CDCl3) δ7.55(d,J=8.4Hz,1H),7.25(d,J=15.7Hz,1H),7.09(d,J=15.7Hz,2H),7.00(d, J=7.3Hz,1H),6.68(s,2H),6.19(d,J=8.3Hz,1H),4.25(d,J=13.1Hz,2H),4.08(s,2H),3.75(s,3H). 13 CNMR(101MHz, CDCl3)δ182.25,159.98,159.87,157.56,155.60,154.66,144.01,142.18,127.97 ,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] Example 10 Synthesis of Substituted Isatin-Coumarin Derivative IC-3c

[0062]

[0063] The difference from Example 1 is that the indigo intermediate of this example uses N-(3-bromopropyl)-5-methoxyinsatin instead of N-(2-bromoethyl)-5-chloroinsatin, and other parameters and operations are referred to Example 1 to obtain a substituted indigo-coumarin derivative IC-3c with a yield of 52.8%.

[0064] 1 H NMR (500MHz, CDCl3) δ7.64(d,J=9.5Hz,1H),7.38(d,J=8.5Hz,1H),7.16(d,J=2.4Hz,1H),7.09(dd,J=8.5,2.5Hz,1H),6.86(d,J=8.6Hz,1H ),6.81(d,J=8.6Hz,1H),6.77(s,1H),6.27(d,J=9.5Hz,1H),4.11(t,J=5.6Hz,2H),3.94(t,J=6.8Hz,2H),3.80(s,3H),2.32-2.19(m,2H). 13C NMR (126MHz, CDCl3) δ184.35,161.56,161.11,158.40,156.56,155.80,144.70,143.35,128.95,12 4.82,117.90,113.43,112.90,112.52,110.96,109.73,101.61,65.62,56.00,37.30,27.06.ESI-MS m / z:380.4[M+H] + .

[0065] Example 11 Synthesis of Substituted Isatin-Coumarin Derivative IC-4c

[0066]

[0067] The difference from Example 1 is that the indigo intermediate of this example uses N-(4-bromobutyl)-5-methoxyinsatin instead of N-(2-bromoethyl)-5-chloroinsatin, and other parameters and operations are referred to Example 1 to obtain a substituted indigo-coumarin derivative IC-4c with a yield of 51.1%.

[0068] 1 H NMR (400MHz, CDCl3) δ7.64(d,J=9.5Hz,1H),7.37(d,J=8.6Hz,1H),7.18-7.13(m,2H),6.88-6.84(m,1H),6.82(dd,J=8.6,2.3Hz ,1H),6.77(d,J=2.1Hz,1H),6.25(d,J=9.5Hz,1H),4.08(d,J=5.1Hz,2H),3.81(s,3H),3.80-3.75(m,2H),1.92(d,J=3.2Hz,4H). 13 CNMR(101MHz, CDCl3)δ183.78,161.95,161.21,158.34,156.53,155.84,144.59,143.43,128.87,124.7 3,118.04,113.19,112.69,112.65,111.11,109.75,101.48,67.60,56.01,39.78,26.30,23.97.ESI-MS m / z:394.4[M+H] + .

[0069] Example 12 Synthesis of Substituted Isatin-Coumarin Derivative IC-5c

[0070]

[0071] The difference from Example 1 is that the indigo intermediate of this example uses N-(5-bromopentyl)-5-methoxyinsatin instead of N-(2-bromoethyl)-5-chloroinsatin, and other parameters and operations are referred to Example 1 to obtain a substituted indigo-coumarin derivative IC-5c with a yield of 52.5%.

[0072] 1 H NMR (500MHz, CDCl3) δ7.64(d,J=9.2Hz,1H),7.36(d,J=8.2Hz,1H),7.14(d,J=9.3Hz,2H),6.82(t,J=8.0Hz,2H),6.77(s,1H), 6.25(d,J=9.3Hz,1H),4.01(s,2H),3.81(s,3H),3.75(d,J=7.2Hz,2H),1.88(s,2H),1.83-1.76(m,2H),1.60(d,J=6.5Hz,2H). 13 CNMR(101MHz, CDCl3)δ183.41,162.09,161.35,158.27,156.56,155.89,144.34,143.54,128.75,124.74,1 18.18,113.04,112.74,112.61,111.21,109.88,101.31,67.88,56.14,40.09,28.46,26.48,23.64.ESI-MS m / z:408.4[M+H] + .

[0073] Example 13 Synthesis of Substituted Isatin-Coumarin Derivative IC-2d

[0074]

[0075] The difference from Example 1 is that the indigo intermediate of this example uses N-(2-bromoethyl)-5-bromoinsatin instead of N-(2-bromoethyl)-5-chloroinsatin, and other parameters and operations are referred to Example 1 to obtain a substituted indigo-coumarin derivative IC-2d with a yield of 48.9%.

[0076] 1 H NMR (400MHz, CDCl3) δ7.82-7.61(m,3H),7.38(s,1H),7.12(s,1H),6.76(s,2H),6.28(s,1H),4.33(s,2H),4.17(d,J=27.6Hz,2H). 13C NMR (101MHz, CDCl3) δ181.75,160.91,160.82,157.74,155.66,149.92,143.24,140.56,129 .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] Example 14 Synthesis of Substituted Isatin-Coumarin Derivative IC-3d

[0078]

[0079] The difference from Example 1 is that the indigo intermediate of this example uses N-(3-bromopropyl)-5-bromoinsatin instead of N-(2-bromoethyl)-5-chloroinsatin, and other parameters and operations are referred to Example 1 to obtain a substituted indigo-coumarin derivative IC-3d with a yield of 45.7%.

[0080] 1 H NMR (400MHz, CDCl3) δ7.74(s,1H),7.67(t,J=8.1Hz,2H),7.40(d,J=8.2Hz,1H),6.88(d,J=8.3Hz,1H),6.8 0(d,J=8.1Hz,2H),6.29(d,J=9.5Hz,1H),4.12(t,J=5.6Hz,2H),3.99(t,J=6.8Hz,2H),2.30-2.25(m,2H). 13 C NMR (101MHz, CDCl3) δ182.07,161.40,161.08,157.58,155.78,149.55,143.32,140.64,129.00 ,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] Example 15 Synthesis of Substituted Isatin-Coumarin Derivative IC-4d

[0082]

[0083] The difference from Example 1 is that the indigo intermediate of this example uses N-(4-bromobutyl)-5-bromoinsatin instead of N-(2-bromoethyl)-5-chloroinsatin, and other parameters and operations are referred to Example 1 to obtain a substituted indigo-coumarin derivative IC-4d with a yield of 49.3%.

[0084] 1 H NMR (500MHz, CDCl3) δ7.73-7.70(m,2H),7.64(d,J=9.5Hz,1H),7.37(d,J=8.6Hz,1H),6.86-6.83(m,1H),6.81(dd,J=8.6,2.4H z,1H),6.78(d,J=2.3Hz,1H),6.26(d,J=9.5Hz,1H),4.07(t,J=5.4Hz,2H),3.82(t,J=6.6Hz,2H),1.93(dd,J=7.8,5.4Hz,4H). 13 C NMR (101MHz, CDCl3) δ182.18,161.89,161.16,157.54,155.86,149.45,143.38,140.60,128.87,12 8.37,118.79,116.67,113.26,112.71,112.51,111.77,101.44,67.53,39.97,26.28,23.93.ESI-MS m / z:443.3[M+H] + .

[0085] Example 16 Synthesis of Substituted Isatin Coumarin Derivative IC-5d

[0086]

[0087] The difference from Example 1 is that the indigo intermediate of this example uses N-(5-bromopentyl)-5-bromoinsatin instead of N-(2-bromoethyl)-5-chloroinsatin, and other parameters and operations are referred to Example 1 to obtain a substituted indigo-coumarin derivative IC-5d with a yield of 38.6%.

[0088] 1H NMR (500MHz, DMSO-d6) δ7.77(dd,J=15.6,8.1Hz,2H),7.69-7.63(m,2H),7.39(d,J=8.3Hz,1H),7.34(t,J=7.5Hz,1H),7.20(d,J=8.4Hz,1H ),5.87(s,1H),4.20(t,J=5.9Hz,2H),3.71(t,J=6.7Hz,2H),1.90-1.82(m,2H),1.69(dd,J=14.2,7.0Hz,2H),1.54(dd,J=14.4,7.3Hz,2H). 13 C NMR (126MHz, DMSO) δ182.71,165.38,162.10,158.20,153.19,150.05,140.19,133.16,127.10,12 4.61,123.24,119.71,116.90,115.68,115.28,113.37,90.93,69.71,27.99,26.69,23.04.ESI-MS m / z:457.3[M+H] + .

[0089] Example 17 Synthesis of Substituted Isatin-Coumarin Derivative IC-3e

[0090]

[0091] The difference from Example 1 is that the indigo intermediate of this example uses N-(3-bromopropyl)-6-chloroindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations are the same as those of Example 1 to obtain a substituted indigo-coumarin derivative IC-3e with a yield of 43.9%.

[0092] 1 H NMR (400MHz, DMSO-d6) δ7.99(d,J=9.5Hz,1H),7.62(d,J=8.6Hz,1H),7.55(d,J=8.0Hz,1H),7.36(d,J=1.6Hz,1H),7.13(dd,J= 8.0,1.7Hz,1H),6.94-6.86(m,2H),6.29(d,J=9.5Hz,1H),4.16(t,J=6.0Hz,2H),3.86(t,J=6.6Hz,2H),2.09(p,J=6.3Hz,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 uses N-(4-bromobutyl)-6-chloroindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations are the same as those of Example 1 to obtain a substituted indigo-coumarin derivative IC-4e 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 from Example 1 is that the indigo intermediate of this example uses N-(5-bromopentyl)-6-chloroindigo instead of N-(2-bromoethyl)-5-chloroindigo, and other parameters and operations are referred to Example 1 to obtain a substituted indigo-coumarin derivative IC-5e with a yield of 47.3%.

[0100] 1 H NMR (400MHz, CDCl3) δ7.63(d,J=9.5Hz,1H),7.55(d,J=8.0Hz,1H),7.36(d,J=8.6Hz,1H),7.10(d,J=8.0Hz,1H),6.91(s,1H),6.85-6.72(m, 2H), 6.24 (d, J = 9.5Hz, 1H), 4.03 (t, J = 6.1Hz, 2H), 3.76 (t, J = 7.2Hz, 2H), 1.94-1.85 (m, 2H), 1.80 (dt, J = 14.9, 7.3Hz, 2H), 1.70-1.49 (m, 2H). 13 C NMR (101MHz, CDCl3) δ181.91,162.14,161.21,158.18,155.89,151.87,144.82,143.41,128.79,126.5 6,123.94,115.92,113.09,112.87,112.55,110.87,101.37,68.08,40.25,28.49,26.92,23.41.ESI-MS m / z:412.8[M+H] + .

[0101] Example 20 Synthesis of Substituted Isatin-Coumarin Derivative IC-3f

[0102]

[0103] The difference from Example 1 is that the indigo intermediate of this example is replaced by N-(3-bromopropyl)-5-bromoinsatin instead of N-(2-bromoethyl)-5-chloroinsatin, 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, and the substituted indigo-coumarin derivative IC-3f is obtained with a yield of 47.8%.

[0104] 1H NMR (400MHz, CDCl3) δ7.71(d,J=1.5Hz,1H),7.66(dd,J=6.7,1.5Hz,1H),7.51(d,J=7.0Hz,1H),6.89(d,J=6.7Hz,1H),6.81(dd,J= 7.0,1.9Hz,1H),6.77(d,J=1.9Hz,1H),6.15(s,1H),4.11(t,J=4.4Hz,2H),3.98(t,J=5.4Hz,2H),2.41(s,3H),2.28-2.23(m,2H). 13 C NMR (101MHz, CDCl3) δ182.12,161.24,157.65,155.13,152.60,149.56,140.65,133.78,128.32,12 5.80,118.72,116.67,113.98,112.21,111.73,109.81,101.46,65.47,37.56,26.92,18.75.ESI-MS m / z:443.2[M+H] + .

[0105] Example 21 Synthesis of Substituted Isatin-Coumarin Derivative IC-4f

[0106]

[0107] The difference from Example 1 is that the indigo intermediate of this example is replaced by N-(4-bromobutyl)-5-bromoinsatin instead of N-(2-bromoethyl)-5-chloroinsatin, 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, and the substituted indigo-coumarin derivative IC-4f is obtained with a yield of 48.1%.

[0108] 1 H NMR (400MHz, CDCl3) δ7.72(d,J=9.4Hz,2H),7.50(d,J=6.9Hz,1H),6.86(dd,J=13.9,6.6Hz ,2H),6.78(s,1H),6.14(s,1H),4.17-4.03(m,2H),3.84(s,2H),2.41(s,3H),1.93(s,4H). 13C NMR (101MHz, CDCl3) δ182.23,161.68,161.29,157.52,155.18,152.62,149.44,140.63,128.31,125.6 7,118.73,116.65,113.70,112.40,112.03,111.84,101.39,67.47,39.98,26.28,23.93,18.74.ESI-MS m / z:457.3[M+H] + .

[0109] Example 22 Synthesis of Substituted Isatin-Coumarin Derivative IC-5f

[0110]

[0111] The difference from Example 1 is that the indigo intermediate of this example is replaced by N-(5-bromopentyl)-5-bromoinsatin instead of N-(2-bromoethyl)-5-chloroinsatin, 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, and the substituted indigo-coumarin derivative IC-5f is obtained with a yield of 39.7%.

[0112] 1 H NMR (400MHz, CDCl3) δ7.72-7.69(m,2H),7.50(d,J=7.0Hz,1H),6.87-6.82(m,2H),6.77(d,J=2.8Hz,1H),6.15(s,1H) ,4.02(t,J=4.9Hz,2H),3.78(t,J=5.7Hz,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 (400MHz, CDCl3) δ182.35,161.90,161.37,157.49,155.21,152.67,149.54,140.59,128.31,125.60,1 18.73,116.57,113.55,112.57,111.92,111.83,101.26,67.95,40.18,29.74,28.52,26.89,23.40.ESI-MS m / z:471.3[M+H] + .

[0113] Example 23 Synthesis of Substituted Isatin-Coumarin Derivative IC-3g

[0114]

[0115] The difference from Example 1 is that the indigo intermediate of this example is replaced by N-(3-bromopropyl)-5-methoxyinsatin instead of N-(2-bromoethyl)-5-chloroinsatin, 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, and a substituted indigo-coumarin derivative IC-3g is obtained with a yield of 53.4%.

[0116] 1 H NMR (400MHz, DMSO-d6) δ7.64(d,J=6.9Hz,1H),7.23(dt,J=7.2,3.6Hz,1H),7.15(d,J=6.9Hz,1H),7.11(d,J=2.0Hz,1H),6.95-6.8 9(m,2H),6.19(s,1H),4.10(t,J=4.8Hz,2H),3.77(s,3H),3.71(t,J=5.2Hz,2H),2.38(s,3H),1.79(ddd,J=11.0,9.3,5.5Hz,2H). 13 C NMR (101MHz, DMSO-d6) δ184.15,162.04,160.60,158.61,156.09,155.17,153.84,144.87,126.85,124. 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 Isatin-Coumarin Derivative IC-4g

[0118]

[0119] The difference from Example 1 is that the indigo intermediate of this example is replaced by N-(4-bromobutyl)-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, and a substituted indigo-coumarin derivative IC-4g is obtained with a yield of 50.2%.

[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-methoxy indigo instead of N-(2-bromoethyl)-5-chloro indigo, 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, and a substituted indigo-coumarin derivative IC-5g 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(101MHz,DMSO-d6)δ184.23,162.15,160.61,158.52,156.09,155.18,153.85,144.90,126.85,124.31, 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 Isatin-Coumarin Derivative IC-3h

[0126]

[0127] The difference from Example 1 is that the indigo intermediate of this example is replaced by N-(3-bromopropyl)-insatin instead of N-(2-bromoethyl)-5-chloroinsatin, 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, and the substituted indigo-coumarin derivative IC-3h is obtained with a yield of 45.2%.

[0128] 1 H NMR (400MHz, DMSO-d6) δ7.63(dd,J=12.6,6.6Hz,2H),7.54(d,J=5.6Hz,1H),7.21(d,J=6.1Hz,1H),7.11( t,J=5.7Hz,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=4.2Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ183.86,161.90,160.57,158.71,155.10,153.85,151.07,138.51,126.87,1 24.86,123.53,118.06,113.55,112.83,111.58,111.02,101.58,66.26,37.05,26.76,18.59.ESI-MS m / z:364.4[M+H] + .

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

[0130]

[0131] The difference from Example 1 is that the indigo intermediate of this example is replaced by N-(4-bromobutyl)-isatin 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, and the substituted indigo-coumarin derivative IC-4h is obtained with a yield of 47.5%.

[0132] 1 H NMR (400MHz, DMSO-d6) δ7.65(ddd,J=7.0,5.5,2.2Hz,2H),7.53(d,J=5.7Hz,1H),7.22(d,J=6.4Hz,1H),7.12(t,J=6.0Hz,1 H),6.93(dd,J=10.2,3.2Hz,2H),6.19(s,1H),4.11(t,J=4.8Hz,2H),3.74(t,J=5.3Hz,2H),2.38(s,3H),1.87-1.74(m,4H). 13 C NMR(101MHz,DMSO-d6)δ183.89,162.06,160.60,158.62,155.17,153.85,151.11,138.55,126.86,1 24.88,123.54,117.98,113.48,112.86,111.53,111.14,101.59,68.29,26.23,23.90,18.59.ESI-MS m / z:378.4[M+H] + .

[0133] Example 28 Synthesis of Substituted Isatin-Coumarin Derivative IC-5h

[0134]

[0135] The difference from Example 1 is that the indigo intermediate of this example is replaced by N-(5-bromopentyl)-isatin 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, and the substituted indigo-coumarin derivative IC-5h is obtained with a yield of 44.8%.

[0136] 1 H NMR(400MHz,DMSO-d6)δ7.59(d,J=43.7Hz,3H),7.16(d,J=34.8Hz,2H),6.93( s,2H),6.19(s,1H),4.06(s,2H),3.70(s,2H),2.39(s,3H),1.94-1.36(m,6H).13 C NMR (101MHz, DMSO-d6) δ183.96,162.16,160.62,158.54,155.19,153.86,151.15,138.64,126.86,124. 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] Experimental Example 1 Inhibitory Effect of Substituted Isatin-Coumarin Derivatives on Monoamine Oxidase-B

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

[0139] (1) Preparation of sample solution:

[0140] Weigh samples separately and dissolve them in dimethylsulfoxide (DMSO) to a concentration of 10 mM, store them in a -20°C refrigerator, and dilute them to the required concentration with phosphate buffer (0.2 mol / L, pH 7.6) before use, so that the final DMSO concentration is less than or equal to 0.5% (v / v).

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

[0142] Monoamine oxidase B was purchased from Sigma. A certain amount of monoamine oxidase B was weighed and diluted with deionized water to a suitable 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. 30 μL of substrate and 10 μL of colorimetric solution were immediately added. After incubation at 37°C for 60 min, the absorbance value was measured at λ=490 nm using an enzyme 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 can be seen from the table, the compounds obtained in the present invention all have different degrees of inhibitory effects on MAO-B, among which compound IC-4g has the strongest inhibitory effect, reaching 89.3%, and its activity is stronger than that of the positive control ladostigil. It is proved that the substituted isatin-coumarin derivatives obtained in the present invention can be used to prepare anti-Alzheimer's disease drugs based on inhibiting MAO-B.

[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 indigo carmine part when the aromatic ring is substituted with an electron-donating group is higher than that of the coumarin part when the aromatic ring is substituted with a methoxy group; the activity is better when the aromatic ring of the coumarin part is substituted with a methoxy group; the chain length between the indigo carmine 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 activity 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 concentrations were all 20 μM. Curcumin and 7-hydroxycoumarin were used as positive controls. The specific steps included:

[0155] 1. Preparation of solution:

[0156] (1) 20 mM pH 7.4 phosphate buffer solution (PBS): weigh 3.618 g Na2HPO4 and 0.6027 g KH2PO4, add 100 mL ultrapure water, wait until the solid is completely dissolved, make up to 200 mL with ultrapure water, and adjust the solution pH to 7.4.

[0157] (2) Aβ 1-42 Protein solution: Dissolve 1 mg of protein in 100 μL of 1% NH4OH solution to a concentration of 2300 μM and store in a -80°C refrigerator until use. Dilute to 40 μM with PBS buffer before use.

[0158] (3) 50 mM glycine-NaOH buffer: Weigh 0.938 g of glycine and dissolve it in 250 mL of ultrapure water. Adjust the pH to 8.50 with 1 mol / L NaOH solution and store in a refrigerator at 4 °C.

[0159] (4) 5 μM Thioflavin T solution (prepared and used immediately): Weigh 2.2 mg of Thioflavin T powder and dissolve it in 689 μL of pH 8.5 glycine-NaOH buffer. Ultrasonicate to completely dissolve the solid and keep it away from light.

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

[0161] 2. Inhibitory activity test:

[0162] Take 10 μL of 40 μM Aβ 1-42 The protein was mixed with 10 μL of a 40 μM compound and placed in an incubator 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; the positive control was Aβ 1-42 Protein was incubated with resveratrol. After 72 hours, the incubation solution was transferred to a black 96-well plate, 180 μL of 5 μM Thioflavin T solution was added, and the reaction was allowed to stand in the dark at room temperature for 5 minutes. Finally, the fluorescence absorption value was measured using a multifunctional microplate reader, with an excitation wavelength of 450 nm and an absorption wavelength of 485 nm. 1-42 The fluorescence intensity of the compound bound to Thioflavin T was used as a control to determine the effect of the compound on Aβ 1-42 The results are shown in Table 2. The results show that the compounds of the present invention have an inhibitory effect on Aβ 1-42The compounds IC-4g and IC-3g showed the strongest inhibitory activity, reaching 88.9% and 83.4%, respectively. This proves that the substituted isatin-coumarin derivatives of the present invention have great development prospects and can be used to inhibit Aβ 1-42 Self-aggregation for the preparation of anti-Alzheimer's disease drugs.

[0163] In addition, according to the compound structure and the overall structure-activity relationship in Table 2, the following may be true: the activity of the isatin and coumarin moieties substituted with electron-donating groups is higher than that of the coumarin moieties substituted with electron-withdrawing groups; the chain length between the isatin moiety and coumarin has an effect on the activity, and the activity is better when the carbon chain length is 4.

[0164] Table 2 Effects of substituted isatin-coumarin derivatives on Aβ 1-42 Self-aggregation inhibitory activity

[0165] Compound <![CDATA[Aβ 1-42 Self-aggregation inhibition rate]]> Compound <![CDATA[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 test of substituted isatin-coumarin derivatives

[0167] The in vitro antioxidant activity of the substituted isatin-coumarin derivatives obtained in Examples 1 to 28 was determined by the ORAC method, with AAPH as the source of peroxyl radicals and sodium fluorescein (FL) as the fluorescent indicator to evaluate the antioxidant capacity of some compounds, and the experimental results were expressed as Trolox equivalents. Specifically, the following steps were included:

[0168] (1) Preparation of phosphate buffered saline (PBS): Weigh an appropriate amount of phosphoric acid and dilute it with ultrapure water to obtain a 75 mM phosphoric acid 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 the phosphoric acid solution to obtain a 75 mM phosphate buffered saline with a pH of 7.4.

[0169] (2) AAPH solution (prepared and used immediately): Accurately weigh 0.0588 g of AAPH and dissolve it in 5.42 mL of phosphate buffer to make up to a concentration of 40.0 mM AAPH solution.

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

[0171] (4) Preparation of Trolox solution: Accurately weigh 2.50 mg of Trolox and dissolve it in 1000 μL DMSO using a pipette to obtain a 10 mM Trolox solution. During the experiment, accurately pipette the Trolox DMSO solution and dilute it to the test concentration using phosphate buffer.

[0172] (5) Preparation of compound solution: Accurately weigh an appropriate amount of compound using a precision analytical balance, dilute with DMSO to a transparent solution with a concentration of 1 mM, and dilute with phosphate buffer to the desired concentration when used.

[0173] (6) Antioxidant activity test: 20 μL of different concentrations of compounds or Trolox and 120 μL of FL diluent were respectively pipetted into a black 96-well culture plate, mixed with a dispenser, incubated at 37°C for 15 min, and then 60 μL of AAPH was quickly added. The fluorescence value was measured and recorded every 1 min using a multifunctional microplate reader, with an excitation wavelength of 485 nm and an emission wavelength of 535 nm for a total of 240 min. The blank control was tested with 20 μL PBS instead of the compound. The area between the curve and the coordinate (AUC) was calculated by integration using ORIGIN software. The protection area of ​​the sample was calculated as: NetAUC = AUC antioxidant–AUC blank. 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 Trolox value equivalent.

[0174] Table 3 In vitro antioxidant activity of substituted isatin-coumarin derivatives

[0175]

[0176] As can be seen from Table 3, some of the compounds obtained in the present invention have good antioxidant effects in vitro, among which the ORAC values ​​of compounds IC-5g and IC-4g at a concentration of 5 μM are even as high as 3 or more, indicating good antioxidant properties. This proves that the substituted isatin-coumarin derivatives obtained in the present invention can be used to prepare anti-Alzheimer's disease drugs based on antioxidant effects.

[0177] Experimental Example 4 Inhibitory Effect of Substituted Isatin-Coumarin Derivatives on Acetylcholinesterase

[0178] The inhibitory effect of the substituted isatin-coumarin derivatives obtained in Examples 1 to 28 on acetylcholinesterase was determined by the Ellman method. The results were expressed as IC 50The values ​​are expressed as Tacrine 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:

[0179] (1) Preparation of drug solution:

[0180] Weigh a certain amount of various samples to be analyzed and dissolve them in dimethyl sulfoxide (DMSO) to a concentration of 10 mM, store them in a -20°C refrigerator, and dilute them to the required concentration with phosphate buffer (0.1 mol / L, pH 8.0) before use, so that the final DMSO concentration is less than or equal to 0.5% (v / v).

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

[0182] Acetylcholinesterase (EC3.1.1.7, from electric ell.) was purchased from Sigma. A certain amount of acetylcholinesterase was weighed and diluted with deionized water to an appropriate activity range.

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

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

[0185] (4) Preparation of color developer stock solution:

[0186] The color developing agent 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) was purchased from Sigma. A certain amount of DTNB was weighed and prepared to 0.01 mol / L with phosphate buffer solution (0.1 mol / L, pH 8.0), and stored at 4°C in the dark.

[0187] (5) Testing:

[0188] Select 6 wells in a 96-well plate, add 10 μL enzyme solution, and 0, 5, 10, 20, 35, 50 μL test compound solution, add 0.1 mol / L pH 8.0 phosphate buffer solution to make the total volume 100 μL, incubate in a 37°C full-wavelength microplate reader for 15 minutes, immediately add 10 μL ATC solution, 10 μL DTNB solution and 80 μL phosphate buffer solution, a total of 100 μL, scan at λ = 412nm for 2 minutes to measure the absorbance change. The experimental results are shown in Table 4, and the results show that most of the compounds described in the present invention have good acetylcholinesterase inhibitory activity, which is better than the parent nucleus 7-hydroxycoumarin. Among them, compounds IC-3a, IC-3d and IC-3e have the best inhibitory activity on acetylcholinesterase, and their IC 50 The values ​​are all less than 10 μM, and can be used to prepare anti-Alzheimer's disease drugs.

[0189] Table 4 Inhibitory activity of compounds on acetylcholinesterase

[0190] Compound <![CDATA[IC 50 forAChE(μM)]]> Compound <![CDATA[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 <![CDATA[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 <![CDATA[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] Not determined

[0192] Experimental Example 5 Metal Complexation Experiment of Substituted Isatin-Coumarin Derivatives

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

[0194] 1. Preparation of solution:

[0195] (1) Compound IC-4g solution: Weigh a certain amount of compound and prepare it to 1 mM with anhydrous ethanol.

[0196] (2) Metal ion solution: Weigh a certain amount of NaCl, KCl, CaCl2, ZnSO4, CuSO4 and FeSO4, prepare them to 10 mM with ultrapure water, and then dilute them to 1 mM with anhydrous ethanol.

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

[0198] Take 6 5mL centrifuge tubes, add 360μL 1mM IC-4g compound solution, then add 360μL 1mM NaCl, KCl, CaCl2, ZnSO4, CuSO4 and FeSO4 solution, and finally add anhydrous ethanol to make the total volume 3000μL, so that the final concentration of compound IC-4g and metal ions are 120μM. The blank control is 360μL 1mM compound solution with anhydrous ethanol added to the same concentration as the compound in the sample. After mixing, place at room temperature for 30min, pour into a quartz cuvette and scan the absorption curve with a UV-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 Figure 1 As can be seen from the figure, the compound IC-4g of the present invention has a strong metal complexing ability and has a strong metal complexing ability for Cu 2+ and Fe 2+ It has good selectivity; other substituted isatin-coumarins with similar structures also have similar effects, which proves that the substituted isatin-coumarin derivatives obtained by the present invention can be used to prepare anti-Alzheimer's disease drugs based on metal complexation.

[0200] Experimental Example 6 Study on the toxicity of substituted isatin-coumarin derivatives to nerve cells

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

[0202] 1. Solution Preparation

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

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

[0205] (3) MTT solution: MTT was prepared into 5 g / L using PBS solution, sterilized by filtration using a 0.22 μM filter membrane, and stored in a refrigerator at 4°C away from light.

[0206] 2. Cultivation of SH-SY5Y Neural Cells

[0207] The neural cell line SH-SY5Y was routinely cultured with DMEM medium in an incubator at 37°C, saturated humidity, 5% CO2 and 95% air, and passaged every 2 to 3 days.

[0208] 3. Neuronal Cytotoxicity Assay

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

[0210] (2) Aspirate the original culture medium and add different concentrations of compound solutions diluted with DMEM medium, 100 μL per well, and set up 5 replicates. Add culture medium instead of compound to the blank and control groups, and place in a 37°C, 5% CO2 incubator for 48 hours; 4 hours before the end of the experiment, add culture medium containing 5 mg / mL MTT to the sample group and control group, 100 μL / well, and continue to culture for 4 hours.

[0211] (3) Discard the supernatant, add 100 μL of DMSO to each well, shake to fully dissolve the product formazan, and measure the absorbance value (OD value) of each well on a full-wavelength microplate reader at a wavelength of 570 nm; cell survival rate (%) of each sample = (OD sample - OD blank) / (OD control - OD blank) × 100%; cell inhibition rate (%) of each sample = 100% - cell survival rate (%) of each sample, plot inhibition rate against concentration, and the concentration with an inhibition rate of 50% is the compound IC 50 The results are shown in Table 5.

[0212] Table 5 Toxicity of substituted isatin-coumarin derivatives to neural cells (SH-SY5Y)

[0213] Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[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 values ​​of the substituted isatin-coumarin derivatives obtained in the present invention on SH-SY5Y cells are 50 The values ​​were all greater than 100 μM, showing low neuronal cytotoxicity. Among them, IC-4g, the compound with the strongest inhibitory activity against MAO-B and Aβ self-aggregation, had an 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 cells 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 specific steps of the neuroprotective effect of induced SH-SY5Y cell injury are as follows:

[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. The 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 good neuroprotective effect on SH-SY5Y cell injury 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 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 light-dark cycle of 12 h. The 60 male SD mice were randomly divided into 6 groups: a) control group (injection of normal saline, intragastric administration of distilled water); b) AD model group (Aβ 1-42, 410pmol / 5μL); c) positive control group (donepezil, 8.0mg / kg); d) high-dose group (IC-4g-H 8.0mg / kg); e) medium-dose group (IC-4g-M 4.0mg / kg); f) low-dose group (IC-4g-L 2.0mg / kg). Aβ 1-42 48 hours after injection, each drug-treated group was gavaged once a day for 14 consecutive days. The control group and AD model group were gavaged with distilled water in the same way. After 14 days of treatment, the passive avoidance test was performed according to the following method.

[0222] (2) Passive avoidance experiment

[0223] The in vivo memory enhancement ability was evaluated by the passive avoidance test in mice, which included two separate trials (a training trial and a test trial 24 hours later). The test apparatus consisted of two compartments of equal size (a light compartment and a dark compartment) separated by a guillotine door, and lighting was provided by a 250lx LED light in a light box. In the training trial, the mice were first placed in the light chamber 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, while the electrical stimulator was activated, and the animal's foot was shocked (24V, 0.5mA). The mice were trained repeatedly for 5 minutes. If the mouse failed to enter the dark compartment within 180 seconds, it was eliminated from the test.

[0224] The test was conducted 24 hours after the training trial. We put the mice into the light compartment again and opened the door. The overall 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 It obviously caused memory impairment in mice. After treatment with donepezil and different concentrations of compound IC-4g (2.0, 4.0, 8.0 mg / kg), the latency time and the average number of errors were significantly reversed compared with the model group. In addition, compound IC-4g prolonged the delay time and reduced the number of errors in a dose-dependent manner. Among them, the 8.0 mg / kg dose of compound IC-4g had a better effect in increasing the latency and reducing the number of errors compared with the donepezil group.

[0226] As can be seen from the above, the substituted isatin-coumarin derivatives obtained in the present invention have good MAO-B inhibitory activity, inhibition of Aβ self-aggregation, inhibition of acetylcholinesterase, antioxidant activity and metal chelation, and have low toxicity to nerve cells and high safety. Among them, compound IC-4g has a good inhibitory effect on Aβ 1-42 Induced SH-SY5Y cell damage has a strong neuroprotective effect; animal experiments show that high-dose compound IC-4g can effectively improve Aβ 1-42 It can be seen that the compound provided by the present invention is very suitable for preparing anti-Alzheimer's disease drugs.

[0227] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A novel substituted isatin-coumarin derivative, characterized in that: Having the structure of formula (I): Wherein, 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 to 4.

2. The novel substituted isatin-coumarin derivative according to claim 1, characterized in that: The R is selected from one or more of hydrogen, 5-halogen, 5-methoxy, and 6-halogen; R1 is selected from hydrogen or methyl, and n is a positive integer of 1 to 4.

3. The novel substituted isatin-coumarin derivative according to claim 1, characterized in that: The substituted isatin-coumarin derivative has any of the following structures:

4. The novel substituted isatin-coumarin derivative according to any one of claims 1 to 3, characterized in that: Also included are pharmaceutically acceptable salts of the substituted isatin-coumarin derivatives.

5. A method for preparing a novel substituted isatin-coumarin derivative as claimed in any one of claims 1 to 4, characterized in that: The synthetic route is as follows: The following steps are involved: S1, compound of formula (II) and Br-(CH2) n -Br undergoes a substitution reaction in an alkaline environment to obtain a compound of formula (III); S2, subjecting the compound of formula (III) obtained in step S1 to a substitution reaction with substituted 7-hydroxycoumarin under alkaline conditions to obtain a compound of formula (I); Wherein, the definitions of R, R1, and n are consistent with those in any one of claims 1-4.

6. Use of the novel substituted isatin-coumarin derivative according to any one of claims 1 to 4 in the preparation of monoamine oxidase-B inhibitors.

7. Use of the novel substituted isatin-coumarin derivative according to any one of claims 1 to 4 in the preparation of acetylcholinesterase inhibitors.

8. Use of the novel substituted isatin-coumarin derivative according to any one of claims 1 to 4 in the preparation of Aβ aggregation inhibitors.

9. Use of the novel substituted isatin-coumarin derivative according to any one of claims 1 to 4 in the preparation of antioxidants.

10. Use of the novel substituted isatin-coumarin derivative according to any one of claims 1 to 4 in the preparation of a metal complexing agent.

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