Betulinic acid derivative containing amide and secondary amine structures as well as preparation method and application of betulinic acid derivative

Abstract

The invention discloses betulinic acid derivatives containing amide and secondary amine structures and a preparation method and application of the betulinic acid derivatives, and belongs to the technical field of medicines.The betulinic acid derivatives are obtained by conducting structural modification on betulinic acid, and it is proved through an in-vitro thioflavin (Th) T fluorescence experiment that the betulinic acid derivatives have a good application prospect. The compounds can effectively inhibit aggregation of beta-amyloid protein (A beta) 42, can be used as candidate drugs for treating Alzheimer's disease, and have good application prospects in clinic.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a betulinic acid derivative containing amide and secondary amine structures, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD), commonly known as "senile dementia", is a chronic neurodegenerative disease caused by damage or decline of the central nervous system. Clinically, AD can be divided into three periods: early, middle and late. Early symptoms include poor judgment, difficulty in object recognition, lack of sense of direction, etc.; mid-term symptoms include memory loss, emotional instability, poor attention, hallucinations, etc.; late symptoms include loss of walking ability, loss of recognition ability, visual impairment, inability to communicate normally, etc. At present, the pathogenic mechanism of AD is still unclear, and there are mainly five hypotheses: Aβ cascade hypothesis, abnormal Tau protein phosphorylation hypothesis, internal cholinergic hypothesis, neuroinflammation hypothesis and metal ion disorder hypothesis. Among them, the more recognized Aβ cascade hypothesis believes that when the Aβ hydrolysis balance is disrupted, a large amount of Aβ will gradually fibrosis after deposition and finally form highly neurotoxic aggregate fragments. Aggregate fragments will continue to combine with more Aβ monomers and other aggregate fragments to form larger fibrous aggregates, becoming one of the main components of senile plaques in the brains of AD patients. In addition, the neurotoxicity of aggregates can damage brain cells, especially nerve cells. When a large number of neurons in the brain are damaged or die, brain function is gradually impaired or even lost, which is an important cause of AD symptoms. Aβ has two main forms: Aβ 40 and Aβ 42 , where Aβ 42 Aβ is more likely to form aggregates and its aggregated 42 Fibrils are key molecules that cause Alzheimer's disease. Therefore, how to inhibit Aβ aggregation, clear Aβ aggregates or reduce Aβ levels has become an important direction for the treatment of AD.

[0003] At present, the classic therapeutic drugs for AD: donepezil, rivastigmine, galantamine, memantine, etc., which are mainly cholinesterase inhibitors and glutamate receptor antagonists, cannot cure the disease, but can only relieve some of its symptoms, and have obvious side effects. Long-term use will lose its efficacy or even aggravate the condition. In addition, aducanumab has problems such as limited therapeutic effect and insufficient clinical experimental data. The therapeutic effect of lencanerumab on AD still needs more clinical data to prove. Therefore, there is an urgent need to develop highly effective and low-toxic AD therapeutic drugs.

[0004] Betulinic acid (BA) is a lupine-type pentacyclic triterpenoid compound, which is widely found in many plants in nature, especially in the bark of Betulaceae plants. Modern pharmacological studies have found that betulinic acid has multiple biological activities such as anti-tumor, anti-inflammatory, antioxidant, anti-HIV, anti-diabetic, and antibacterial. In addition, studies have shown that betulinic acid has a good preventive and therapeutic effect on AD, can effectively inhibit Aβ aggregation or clear Aβ aggregates, inhibit the activity of cholinesterase and glutaminase, reduce oxidative stress response and the production of neuroinflammatory mediators, and has the function of improving cognition and protecting nerves. However, due to the poor water solubility of betulinic acid and its poor solubility in most organic solvents, a higher drug concentration is required for the treatment of diseases and the half-life in vivo is short, which greatly reduces its bioavailability in the body and seriously affects its biological activity.

[0005] Based on this, a method for appropriate structural modification of betulinic acid is proposed to obtain betulinic acid derivatives with novel structure, high bioavailability and strong biological activity. Summary of the invention

[0006] The purpose of the present invention is to provide a betulinic acid derivative containing amide and secondary amine structures and a preparation method and application thereof, so as to solve the problems in the background technology.

[0007] To achieve the above object, the present invention provides a betulinic acid derivative containing an amide and a secondary amine structure, wherein the betulinic acid derivative is one of compound 1 or compound 2, and the structural formula of compound 1 is:

[0008]

[0009] The structural formula of compound 2 is:

[0010]

[0011] Wherein, R is an alkyl group, a heteroalkyl group, an aryl group or a heteroaryl group.

[0012] Preferably, in the structural formula of the compound 1, R is one of the following groups:

[0013]

[0014] Preferably, in the structural formula of the compound 2, R is one of the following groups:

[0015]

[0016] Preferably, the structural formula of the betulinic acid derivative is one of the following compounds I-1a to I-2i:

[0017]

[0018]

[0019] The present invention also provides a method for preparing a betulinic acid derivative, wherein the synthetic route of the compound 1 is:

[0020]

[0021] The synthetic route of the compound 2 is:

[0022]

[0023] Preferably, the specific preparation steps of the compound 1 are:

[0024] S1. Preparation of compound BA-1: Place betulinic acid in a round-bottom flask, add pyridine to dissolve, then add acetic anhydride, and react at room temperature; adjust the pH of the reaction mixture with 3.00 mol / L hydrochloric acid solution, and stir and mix at room temperature; then place the mixed solution in a separatory funnel, add ethyl acetate to dilute, wash the organic layer with distilled water, then wash with saturated sodium chloride solution, dry the organic layer with anhydrous sodium sulfate, concentrate, and then purify by column chromatography to obtain compound BA-1;

[0025] S2. Preparation of the compound represented by formula (III): Compound BA-1 is placed in two round-bottom flasks, dichloromethane is added to dissolve under nitrogen protection, and oxalyl chloride is slowly added under ice bath conditions. After a period of reaction, the crude acyl chloride is directly spin-dried to obtain a crude acyl chloride; the compound represented by formula (II) is placed in a round-bottom flask, dichloromethane is added to dissolve, and triethylamine is added to obtain an amine mixed solution; dichloromethane is added to the crude acyl chloride obtained above to dissolve to prepare a solution, and the solution is added dropwise to the amine mixed solution under ice bath conditions, and reacted at room temperature; the pH of the reaction mixture is adjusted with 1.00 mol / L hydrochloric acid solution, dichloromethane is added to dilute, the organic layer is washed with distilled water, and then with a saturated sodium chloride solution, the organic layer is dried over anhydrous sodium sulfate, and concentrated to obtain the compound represented by formula (III);

[0026] S3. Preparation of compound 1: Place the prepared compound of formula (III) in a round-bottom flask, add methanol and tetrahydrofuran to dissolve it, then add sodium hydroxide solution and react at room temperature; after the reaction, use 1.00 mol / L hydrochloric acid solution to adjust the pH of the reaction solution, place the mixed solution in a separatory funnel, add ethyl acetate to extract the aqueous layer, combine the organic layers, wash the organic layers with saturated sodium chloride solution, dry the organic layers with anhydrous sodium sulfate, concentrate and purify by column chromatography to obtain the compound of formula I-1.

[0027] Preferably, the specific preparation steps of the compound 2 are:

[0028] S1. Preparation of compound BA-2: Betulin was placed in a 200 mL round-bottom flask, and dichloromethane was added to dissolve it; diacetoxyiodobenzene was added under ice bath conditions, and then TEMPO was added to react at room temperature; after the reaction, a saturated sodium thiosulfate solution was added, and the mixture was stirred and mixed, and then placed in a separatory funnel, and the aqueous layer was extracted with dichloromethane, and the organic layers were combined, and the organic layers were washed with a saturated sodium chloride solution, and the organic layers were dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain compound BA-2;

[0029] S2. Preparation of compound 2: Compound BA-2 was placed in a two-mouth round-bottom flask, 1,2-dichloroethane was added under nitrogen protection to dissolve, and then the compound represented by formula (II) and acetic acid were added in sequence. After a period of reaction, the reaction solution was moved to an ice bath, methanol was added, and then sodium cyanoborohydride was added, and the reaction was carried out at room temperature for 4 hours. After the reaction was completed, a saturated sodium bicarbonate solution was added, and the mixed solution was placed in a separatory funnel, the aqueous layer was extracted with dichloromethane, the organic layers were combined, the organic layers were washed with a saturated sodium chloride solution, the organic layers were dried with anhydrous sodium sulfate, and the concentrated organic layers were purified by column chromatography to obtain the compound represented by formula I-2.

[0030] The present invention also provides the use of the betulic acid derivatives. The betulic acid derivatives prepared by the above preparation method are used in the treatment of Alzheimer's disease, inhibiting Aβ 42 of gathering.

[0031] Therefore, the present invention provides a betulinic acid derivative containing an amide and a secondary amine structure, and a preparation method and application thereof. By structurally modifying betulinic acid, a plurality of betulinic acid derivatives are obtained, and it is confirmed by an in vitro thioflavin (Th) T fluorescence experiment that these compounds can effectively inhibit the expression of β-amyloid protein (Aβ). 42 The aggregation of proteins can be used as candidate drugs for the treatment of Alzheimer's disease and has good application prospects in clinical practice.

[0032] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a graph showing the ratio of ThT fluorescence intensity of the betulinic acid derivatives prepared in Examples 1 to 21 of the present invention at different culture times. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0036] Example 1

[0037] Preparation of compound Ⅰ-1a:

[0038]

[0039] The structural formula of the intermediate compound III-1a is:

[0040]

[0041] The specific preparation steps are as follows:

[0042] S1. Preparation of compound BA-1: Betulinic acid (2.00 g, 4.38 mmol) was placed in a 200 mL round-bottom flask, and pyridine (50.00 mL) was added to dissolve, and acetic anhydride (6.15 mL, 65.70 mmol) was added, and the reaction was carried out at room temperature for 8 h. The reaction mixture was adjusted to pH 5-6 using 3.00 mol / L hydrochloric acid solution, and stirred at room temperature for 30 min. The mixed solution was placed in a separatory funnel, ethyl acetate (150.00 mL) was added to dilute, the organic layer was washed with distilled water (2×100.00 mL), and then washed with saturated sodium chloride solution (1×100.00 mL), the organic layer was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain compound BA-1 (white solid, yield 82%). 1 H NMR(500MHz, CDCl3)δ10.04(br,1H),4.74(s,1H),4.61(s,1H),4.49-4.46(m,1H), 3.00(t,1H,J=13.3Hz),2.28-2.16(m,2H),2.04(s,3H),2.01-1.94(m,2H),1.71-1 .69(m,4H),1.63-1.59(m,3H),1.55-1.47(m,3H),1.44-1.38(m,6H),1.32-1.23(m ,3H),1.20-1.17(m,1H),1.07-0.93(m,8H),0.85-0.83(m,9H),0.80-0.78(m,1H).

[0043] S2. Preparation of compound III-1a: Compound BA-1 (50.00 mg, 0.10 mmol) was placed in a 25 mL two-necked round-bottom flask, and dichloromethane (6.00 mL) was added under nitrogen protection to dissolve, and oxalyl chloride (42.59 μL, 0.50 mmol) was slowly added under ice bath conditions, and the reaction was carried out at 40°C for 4 hours, and then the crude acyl chloride was directly dried. Cyclohexylmethylamine (39.49 μL, 0.30 mmol) was placed in a 25 mL round-bottom flask, and dichloromethane (2.00 mL) was added to dissolve, and then triethylamine (41.68 μL, 0.30 mmol) was added to obtain a cyclohexylmethylamine mixed solution. Dichloromethane (5.00 mL) was added to the above-mentioned crude acyl chloride to dissolve, and it was added dropwise to the mixed solution of cyclohexylmethylamine under ice bath conditions, and reacted at room temperature for 3 hours. The reaction mixture was adjusted to pH 5-6 using 1.00 mol / L hydrochloric acid solution, and diluted with dichloromethane (25.00 mL). The organic layer was washed with distilled water (2×25.00 mL), and then with saturated sodium chloride solution (1×25.00 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to obtain a crude product of compound III-1a. 1 H NMR(300MHz, CDCl3)δ5.65(t,1H,J=6.0Hz),4.73-4.72(m,1H),4.59-4.57(m,1H),4.49-4.43(m,1 H),3.19-3.10(m,2H),3.03-2.94(m,1H),2.45(td,1H,J=12.3,3.6Hz),2.03(s,3H),2.00-1.88(m ,2H),1.76-1.72(m,3H),1.69-1.68(m,7H)1.63-1.59(m,3H),1.56-1.47(m,4H),1.44-1.33(m,6H ),1.31-1.24(m,4H),1.21-1.11(m,4H),1.01-0.93(m,9H),0.84-0.82(m,9H),0.79-0.76(m,1H).

[0044] S3. Preparation of compound Ⅰ-1a: The crude compound Ⅲ-1a obtained above was placed in a 25mL round-bottom flask, methanol (2.00mL) and tetrahydrofuran (4.00mL) were added to dissolve, and then 4.00mol / L sodium hydroxide solution (1.50mL, 6.00mmol) was added, and the reaction was carried out at room temperature for 8h. After the reaction, 1.00mol / L hydrochloric acid solution was used to adjust the pH of the reaction solution to 7-8, the mixed solution was placed in a separatory funnel, ethyl acetate (3×20.00mL) was added to extract the water layer, the organic layers were combined, and the organic layers were washed with saturated sodium chloride solution (1×30.00mL), and the organic layers were dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain compound Ⅰ-1a (white solid, yield 92%). 1 HNMR(300MHz, CDCl3)δ5.65(t,1H,J=6.0Hz),4.73-4.72(m,1H),4.58-4.57(m,1H),3.20-3.10(m ,3H),3.02-2.93(m,1H),2.45(td,1H,J=12.3,3.6Hz),2.05-1.85(m,2H),1.76-1.72(m,3H),1.6 9-1.64(m,8H),1.60-1.55(m,3H),1.51-1.47(m,3H),1.43-1.33(m,6H),1.31-1.24(m,4H),1.21 -1.11(m,4H),1.00-0.95(m,7H),0.93-0.87(m,5H),0.81(s,3H),0.74(s,3H),0.69-0.65(m,1H).

[0045] Example 2

[0046] Preparation of compound Ⅰ-1b:

[0047]

[0048] The structural formula of the intermediate compound III-1b is:

[0049]

[0050] The specific preparation steps are the same as those in Example 1, except that the formula (II) used in S2 is replaced with isopropanolamine to prepare a crude compound III-1b. 1H NMR(300MHz, CDCl3)δ6.01(s,1H),4.74-4.73(m,1H),4.60-4.59(m,1H),4.49-4.44(m,1H),3.95-3.89(m,1H) ,3.52-3.34(m,1H),3.25-3.04(m,2H),2.50-2.38(m,1H),2.04(s,3H),1.97-1.89(m,2H),1.80-1.73(m,1H), 1.68(s,4H),1.64(s,1H),1.61-1.58(m,3H),1.54-1.48(m,3H),1.42-1.40(m,2H),1.38-1.34(m,3H),1.27-1 .25(m,4H),1.20-1.17(m,4H),1.14-1.07(m,1H),0.95(d,6H,J=6.4Hz),0.83-0.82(m,9H),0.80-0.76(m,1H).

[0051] The prepared compound Ⅰ-1b was a white solid with a yield of 86%; 1 H NMR(300MHz, CDCl3)δ6.05(s,1H),4.73-4.72(m,1H),4.59-4.58(m,1H),3.94-3.88(m,1H) ,3.51-3.33(m,1H),3.23-3.04(m,3H),2.50-2.38(m,1H),1.99-1.88(m,2H),1.80-1.73(m 1H),1.68(s,4H),1.61-1.53(m,4H),1.50-1.47(m,2H),1.43-1.32(m,7H),1.28-1.25(m,2H),1.18( d,4H,J=6.2Hz),0.96-0.93(m,10H),0.89-0.85(m,1H),0.81(s,3H),0.74(s,3H),0.69-0.60(m,1H).

[0052] Example 3

[0053] Preparation of compound Ⅰ-1c:

[0054]

[0055] The structural formula of the intermediate compound III-1c is:

[0056]

[0057] The specific preparation steps are the same as those in Example 1, except that the formula (II) used in S2 is replaced with (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamic acid tert-butyl ester to obtain a crude compound III-1c. 1H NMR (300MHz, CDCl3) δ6.05 (s, 1H), 5.00 (s, 1H), 4.72 (s, 1H), 4.58 (s, 1H), 4.48-4.43 (m, 1H), 3.61-3.59 (m, 4H), 3.55-3.52 (m, 4H), 3.49-3.37 (m, 2H), 3.35-3.30 (m, 2H), 3.16-3.07 (m, 1H), 2.43 (t, 1H, J = 11.9 Hz), 2.03 (s, 3H), 1.98-1.87 (m, 2H), 1. 78-1.71(m,1H),1.67(s,4H),1.63-1.58(m,2H),1.55-1.50(m,2H),1.44-1.41(m,12H),1.37-1.33(m,3H),1.26-1.23(m,3H ),1.20-1.07(m,2H),1.01-0.96(m,1H),0.95(s,3H),0.92(s,3H),0.87-0.84(m,2H),0.83-0.81(m,9H),0.79-0.75(m,1H).

[0058] The obtained compound Ⅰ-1c was a white solid with a yield of 89%; 1H NMR (300MHz, CDCl3) δ6.06 (s, 1H), 5.02 (s, 1H), 4.72 (s, 1H), 4.57 (s, 1H), 3.59-3.52 (m, 8H), 3.50-3.36 (m, 2H), 3.35-3.28 (m, 2H), 3.19-3.08 (m, 2H), 2.42 (t, 1H, J = 11.8 Hz), 2.03-1.87 (m, 2H), 1.78-1.69 (m ,2H),1.67(s,4H),1.60-1.50(m,5H),1.47-1.42(m,11H),1.39-1.33(m,5H),1.30-1.24(m,3H),1.15- 1.10(m,1H),0.95(s,6H),0.92(s,3H),0.87-0.84(m,1H),0.80(s,3H),0.74(s,3H),0.68-0.59(m,1H).

[0059] Example 4

[0060] Preparation of compound Ⅰ-1d:

[0061]

[0062] The structural formula of compound III-1d is:

[0063]

[0064] The specific preparation steps are the same as those in Example 1, except that the formula (II) used in S2 is replaced with tert-butyl (3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamate to obtain a crude compound III-1d. 1 H NMR (300MHz, CDCl3) δ6.22(s,1H),4.94(s,1H),4.71(s,1H),4.56(s,1H),4.47-4.42(m,1H),3.64-3.60(m,6H),3.5 7-3.50(m,6H),3.40-3.23(m,2H),3.21-3.08(m,3H),2.45(td,1H,J=12.3,3.5Hz),2.02(s,3H),1.94-1.83(m,2H), 1.80-1.69(m,6H),1.66(s,3H),1.62-1.54(m,3H),1.52-1.45(m,3H),1.42-1.38(m,13H),1.36-1.30(m,4H),1.29- 1.19(m,3H),1.15-1.07(m,1H),1.04-0.98(m,1H),0.93(s,3H),0.91(s,3H),0.87-0.81(m,8H),0.78-0.74(m,1H).

[0065] The prepared compound Ⅰ-1d was a white solid with a yield of 88%; 1 H NMR(300MHz, CDCl3)δ6.23(s,1H),4.95(s,1H),4.70(s,1H),4.55(s,1H),3.62-3.49(m,12H),3.41-3.2 6(m,2H),3.23-3.08(m,4H),2.43(td,1H,J=12.4,3.6Hz),2.07-1.86(m,2H),1.79-1.69(m,5H),1.65-1. 61(m,5H),1.58-1.55(m,2H),1.51-1.45(m,5H),1.41-1.37(m,10H),1.34-1.29(m,4H),1.26-1.22(m,3H ),1.10-1.07(m,1H),0.93-0.91(m,9H),0.85-0.83(m,1H),0.79(s,3H),0.72(s,3H),0.66-0.58(m,1H).

[0066] Example 5

[0067] Preparation of compound Ⅰ-1e:

[0068]

[0069] The structural formula of compound III-1e is:

[0070]

[0071] The specific preparation steps are the same as those in Example 1, except that the formula (II) used in S2 is replaced with 3,5-difluorobenzylamine to obtain a crude product of compound III-1e. 1 H NMR (300MHz, CDCl3) δ6.84-6.78(m,2H),6.70(tt,1H,J=9.1,2.4Hz),5.98(t,1H,J=5.9Hz),4.74(s,1H),4.60(s,1H ),4.51-4.27(m,3H),3.14(td,1H,J=11.0,4.0Hz),2.45(td,1H,J=12.7,3.5Hz),2.04(s,3H),1.98-1.91(m,2H),1.8 0-1.73(m,1H),1.69(s,4H),1.64-1.59(m,3H),1.56-1.53(m,2H),1.50-1.46(m,2H),1.43-1.41(m,3H),1.38-1.33 (m,3H),1.28-1.21(m,4H),1.17-1.13(m,1H),1.01-0.94(m,4H),0.88(s,3H),0.84-0.83(m,8H),0.79-0.76(m,1H).

[0072] The prepared compound Ⅰ-1e was a white solid with a yield of 81%; 1H NMR (300MHz, CDCl3) δ6.82-6.79 (m, 2H), 6.70 (tt, 1H, J = 8.9, 2.3Hz), 5.99 (t, 1H, J = 6.1Hz), 4.73 (s, 1H), 4.59 (s, 1H) ,4.51-4.28(m,2H),3.21-3.10(m,2H),2.44(td,1H,J=12.4,3.6Hz),2.00-1.88(m,2H),1.80-1.73(m,1H),1.68(s,4H ),1.64-1.62(m,1H),1.60-1.57(m,3H),1.55-1.52(m,1H),1.49-1.45(m,2H),1.42-1.41(m,3H),1.39-1.34(m,4H),1 .26-1.24(m,3H),1.19-1.12(m,1H),0.96(d,6H,J=2.2Hz),0.88(s,3H),0.81(s,3H),0.75(s,3H),0.69-0.65(m,1H).

[0073] Example 6

[0074] Preparation of compound Ⅰ-1f:

[0075]

[0076] The structural formula of compound III-1f is:

[0077]

[0078] The specific preparation steps are the same as those in Example 1, except that the formula (II) used in S2 is replaced with R-(+)-1-phenylethylamine to obtain a crude product of compound III-1f. 1H NMR (300MHz, CDCl3) δ7.33-7.27(m,4H),7.29-7.22(m,1H),5.75(d,1H,J=7.6Hz), 5.08(p,1H,J=7.1Hz), 4.72-4.71(m,1H),4.59-4.57(m,1H),4.48-4.43(m,1H),3.20-3.12(m,1H),2.37(td,1H,J=12.4,3.7Hz), 2.03(s,3H),1.99-1.88(m,2H),1.79-1.73(m,1H),1.67(s,4H),1.60-1.54(m,5H),1.48-1.45(m,4H),1. 39-1.34(m,4H),1.25-1.22(m,6H),1.08-1.03(m,2H),0.94-0.92(m,4H),0.83-0.80(m,9H),0.70(s,3H).

[0079] The prepared compound Ⅰ-1f was a white solid with a yield of 90%; 1 H NMR (300MHz, CDCl3) δ7.33-7.30(m,4H),7.29-7.22(m,1H),5.76(d,1H,J=7.6Hz),5.08(p,1H,J=7.1Hz),4.72-4 .71(m,1H),4.58-4.57(m,1H),3.20-3.11(m,2H),2.37(td,1H,J=12.3,3.6Hz),2.02-1.87(m,2H),1.79-1.73(m ,1H),1.67(s,4H),1.59-1.53(m,3H),1.49-1.45(m,4H),1.42(s,2H),1.36-1.24(m,7H),1.22-1.18(m,2H),1.0 9-1.01(m,1H),0.98-0.93(m,7H),0.87-0.85(m,1H),0.78(s,3H),0.74(s,3H),0.70(s,3H),0.66-0.57(m,1H).

[0080] Example 7

[0081] Preparation of compound Ⅰ-1g:

[0082]

[0083] The structural formula of compound III-1g is:

[0084]

[0085] The specific preparation steps are the same as those in Example 1, except that the formula (II) used in S2 is replaced with 2-furanmethylamine to obtain a crude product of compound III-1 g. 1 H NMR (300MHz, CDCl3) δ7.34-7.33(m,1H),6.32-6.30(m,1H),6.22-6.21(m,1H),5.88(t,1H,J=5.6Hz),4.74-4.73( m,1H),4.59-4.58(m,1H),4.53-4.30(m,3H),3.19-3.10(m,1H),2.42(td,1H,J=12.4,3.6Hz),2.03(s,3H),1.95-1 .88(m,2H),1.77-1.73(m,1H),1.68(s,4H),1.61-1.60(m,2H),1.56-1.51(m,2H),1.44-1.42(m,1H),1.38-1.35(m ,2H),1.28-1.25(m,7H),1.13-1.11(m,1H),1.09-1.07(m,1H),0.94(s,4H),0.88-0.86(m,5H),0.83-0.82(m,9H).

[0086] The prepared compound Ⅰ-1g was a white solid with a yield of 83%; 1 H NMR (300MHz, CDCl3) δ7.34-7.33(m,1H),6.32-6.30(m,1H),6.22-6.21(m,1H),5.88(t,1H,J=5.6Hz),4.74- 4.73(m,1H),4.59-4.58(m,1H),4.53-4.30(m,2H),3.20-3.11(m,2H),2.42(td,1H,J=12.4,3.6Hz),2.04-1. 88(m,2H),1.77-1.70(m,1H),1.67(s,4H),1.60-1.55(m,3H),1.52-1.50(m,1H),1.42-1.30(m,8H),1.25-1 .23(m,4H),1.14-1.07(m,1H),0.95(s,6H),0.88-0.86(m,4H),0.81(s,3H),0.75(s,3H),0.68-0.64(m,1H).

[0087] Example 8

[0088] Preparation of compound Ⅰ-1h:

[0089]

[0090] The chemical formula of compound III-1h is:

[0091]

[0092] The specific preparation steps are the same as those in Example 1, except that the formula (II) used in S2 is replaced with histamine to obtain a crude product of compound III-1h. 1 H NMR (300MHz, CDCl3) δ7.87(s,1H),6.89(s,1H),6.58(s,1H),4.72(s,1H),4.59(s,1H),4.48- 4.43(m,1H),3.60-3.54(m,2H),3.08(t,1H,J=9.5Hz),2.88(t,2H,J=6.4Hz),2.47-2.39(m,1H ),2.03(s,3H),1.98-1.82(m,2H),1.67(s,5H),1.63-1.55(m,4H),1.46-1.41(m,3H),1.38-1. 32(m,5H),1.25(s,4H),1.11-1.07(m,2H),0.98-0.94(m,4H),0.88(s,3H),0.83-0.82(m,9H).

[0093] The prepared compound Ⅰ-1h was a white solid with a yield of 78%; 1 H NMR (300MHz, CDCl3) δ7.57(s,1H),6.82(s,1H),6.49(s,1H),4.72(s,1H),4.58(s,1H),3.57-3.49 (m,2H),3.21-3.07(m,2H),2.81(t,2H,J=6.5Hz),2.43(td,1H,J=12.1,2.9Hz),1.99-1.83(m,2H), 1.74-1.67(m,5H),1.61-1.59(m,1H),1.56-1.49(m,3H),1.45-1.31(m,7H),1.28-1.18(m,4H),1.1 1-1.05(m,1H),1.01-0.95(m,7H),0.88-0.85(m,4H),0.80(s,3H),0.75(s,3H),0.68-0.65(m,1H).

[0094] Example 9

[0095] Preparation of compound Ⅰ-1i:

[0096]

[0097] The structural formula of compound III-1i is:

[0098]

[0099] The specific preparation steps are the same as those in Example 1, except that the formula (II) used in S2 is replaced with tryptamine to obtain a crude compound III-1i. 1 H NMR (300MHz, CDCl3) δ8.07 (s, 1H), 7.63 (d, 1H, J = 7.8Hz), 7.40-7.36 (m, 1H), 7.24-7.19 (m, 1H), 7.16-7.10 (m, 1H), 7.04 (d, 1H, J = 2.3Hz), 5.6 4(t,1H,J=5.7Hz),4.71-4.70(m,1H),4.59-4.57(m,1H),4.49-4.43(m ,1H),3.72-3.50(m,2H),3.10-2.90(m,3H),2.37(td,1H,J=12.4,3.6Hz ),2.04(s,3H),1.95-1.87(m,1H),1.81-1.76(m,1H),1.68-1.66(m,4H ),1.63-1.61(m,3H),1.51-1.46(m,2H),1.43(s,3H),1.37-1.32(m,4H) ,1.27-1.23(m,4H),1.16-1.12(m,1H),1.03-0.97(m,2H),0.91(s,3H) ,0.89-0.88(m,1H),0.85(s,3H),0.83-0.82(m,8H),0.78-0.74(m,1H).

[0100] The prepared compound Ⅰ-1i was a white solid with a yield of 87%; 1H NMR (300MHz, CDCl3) δ8.10 (s, 1H), 7.63 (d, 1H, J = 7.8Hz), 7.40-7.37 (m, 1H), 7.24-7.19 (m, 1H), 7.16-7.10 (m, 1H), 7.04 (d, 1H, J = 2.3Hz), 5.64 (t ,1H,J=5.6Hz),4.71-4.70(m,1H),4.58-4.57(m,1H),3.73-3.50(m,2H) ,3.20-3.15(m,1H),3.10-2.90(m,3H),2.37(td,1H,J=12.4,3.6Hz),2.0 1-1.77(m,2H),1.69-1.66(m,4H),1.61-1.60(m,2H),1.53-1.48(m,2H) ,1.44-1.43(m,2H),1.39-1.38(m,2H),1.36-1.31(m,4H),1.27-1.26(m, 2H),1.22-1.19(m,2H),1.03-0.98(m,2H),0.94(d,6H,J=12.1Hz),0.88 -0.86(m,1H),0.85(s,3H),0.80(s,3H),0.75(s,3H),0.67-0.63(m,1H).

[0101] Example 10

[0102] Preparation of compound Ⅰ-1j:

[0103]

[0104] The structural formula of compound III-1j is:

[0105]

[0106] The specific preparation steps are the same as those in Example 1, except that the formula (II) used in S2 is replaced with 3,4-methylenedioxybenzylamine to obtain a crude compound III-1j. 1H NMR (300MHz, CDCl3) δ6.77-6.74(m,3H),5.95(s,2H),5.81(t,1H,J=5.8Hz),4.74(s,1H),4.60(s,1H),4.49 -4.23(m,3H),3.21-3.13(m,1H),2.51-2.42(m,1H),2.04(s,3H),1.97-1.87(m,2H),1.77-1.73(m,1H),1.6 8(s,4H),1.64-1.61(m,3H),1.57-1.56(m,2H),1.51-1.47(m,1H),1.42(s,5H),1.38-1.35(m,2H),1.26(s, 3H),1.14-1.09(m,1H),1.01-0.98(m,1H),0.95(s,3H),0.90(s,3H),0.88-0.87(m,1H),0.84-0.83(m,9H).

[0107] The prepared compound Ⅰ-1j was a white solid with a yield of 79%; 1 H NMR (300MHz, CDCl3) δ6.77-6.74(m,3H),5.94(s,2H),5.83(t,1H,J=5.8Hz),4.74(s,1H),4.59(s,1H) ,4.42-4.22(m,2H),3.21-3.12(m,2H),2.47(td,1H,J=12.6,3.5Hz),2.06-1.86(m,2H),1.77-1.73(m, 1H),1.68(s,4H),1.61-1.53(m,4H),1.51-1.47(m,2H),1.45-1.32(m,7H),1.28(s,1H),1.25-1.22(m ,2H),1.16-1.10(m,1H),0.96(s,6H),0.90-0.88(m,4H),0.82(s,3H),0.75(s,3H),0.69-0.65(m,1H).

[0108] Embodiment 11

[0109] Preparation of Compound I-1k:

[0110]

[0111] The chemical formula of compound III-1k is:

[0112]

[0113] The specific preparation steps are the same as those in Example 1, except that the formula (II) used in S2 is replaced with 2-fluorophenylhydrazine to obtain a crude compound III-1k. 1 H NMR (300MHz, CDCl3) δ7.42 (d, 1H, J = 4.0Hz), 7.05-6.82 (m, 4H), 6.24-6.23 (m, 1H), 4.71-4.70 (m, 1H), 4.59-4.58 ( m,1H),4.49-4.44(m,1H),3.11-3.04(m,1H),2.35(td,1H,J=12.3,3.6Hz),2.09-2.04(m,4H),1.96-1.84(m,2H),1 .67(s,4H),1.63-1.60(m,3H),1.56-1.52(m,3H),1.44-1.43(m,2H),1.38-1.34(m,3H),1.29-1.25(m,4H),1.20-1 .12(m,1H),1.00-0.96(m,4H),0.90-0.88(m,1H),0.84(s,3H),0.83-0.82(m,6H),0.81(s,3H),0.78-0.76(m,1H).

[0114] The prepared compound Ⅰ-1k was a reddish brown solid with a yield of 71%; 1 H NMR (300MHz, CDCl3) δ7.58-7.49(m,2H),7.31-7.18(m,2H),4.78-4.77(m,1H),4.64-4.63(m,1H),3.22-3 .05(m,2H),2.38(td,1H,J=12.6,3.6Hz),2.29-2.20(m,1H),2.12-1.99(m,2H),1.72(s,4H),1.68-1.65(m ,1H),1.62-1.56(m,3H),1.54-1.48(m,3H),1.45-1.42(m,2H),1.40-1.38(m,2H),1.30-1.25(m,4H),1.2 1-1.16(m,2H),1.00(s,6H),0.96(s,3H),0.90-0.87(m,1H),0.84(s,3H),0.76(s,3H),0.71-0.63(m,1H).

[0115] Example 12

[0116] Preparation of compound Ⅰ-11:

[0117]

[0118] The chemical formula of compound III-11 is:

[0119]

[0120] The specific preparation steps are the same as those in Example 1, except that the formula (II) used in S2 is replaced with isoniazid to obtain a crude product of compound III-11. 1 H NMR (300MHz, CDCl3) δ9.22 (s, 1H), 8.75 (d, 2H, J = 5.6Hz), 8.37 (s, 1H), 7.65 (d, 2H, J = 5.9Hz), 4.74 (s, 1H), 4 .62(s,1H),4.49-4.44(m,1H),3.09-3.02(m,1H),2.44-2.35(m,1H),2.13-2.09(m,1H),2.04(s,3H),1.98- 1.90(m,2H),1.73-1.70(m,5H),1.65-1.64(m,2H),1.61-1.60(m,1H),1.57(s,3H),1.52-1.48(m,2H),1.38 (s,3H),1.29-1.25(m,5H),1.21-1.17(m,1H),0.97(d,6H,J=8.5Hz),0.84-0.83(m,9H),0.80-0.77(m,1H).

[0121] The prepared compound Ⅰ-11 was a white solid with a yield of 85%; 1 H NMR (300MHz, CDCl3) δ10.02 (s, 1H), 8.68 (d, 2H, J = 6.1Hz), 8.53 (s, 1H), 7.64 (d, 2H, J = 6.2Hz), 4.7 1(s,1H),4.60(s,1H),3.21-3.15(m,1H),3.06-2.97(m,1H),2.43(td,1H,J=12.7,3.9Hz),2.21-2. 13(m,1H),2.00-1.85(m,2H),1.68(s,6H),1.63(s,1H),1.61-1.56(m,2H),1.53-1.52(m,1H),1.46 -1.34(m,6H),1.31-1.19(m,5H),1.03-0.93(m,10H),0.80(s,3H),0.75(s,3H),0.69-0.66(m,1H).

[0122] Embodiment 13

[0123] Preparation of compound Ⅰ-2a:

[0124]

[0125] The specific preparation steps are:

[0126] S1. Preparation of compound BA-2: Betulin (2.00 g, 4.52 mmol) was placed in a 200 mL round-bottom flask and dissolved in dichloromethane (80.00 mL). Diacetoxyiodobenzene (2.91 g, 9.04 mmol) was added under ice bath conditions, and then TEMPO (142.21 mg, 0.91 mmol) was added, and the mixture was reacted at room temperature for 6 h. After the reaction was completed, a saturated sodium thiosulfate solution (60.00 mL) was added and stirred for 30 min. The mixed solution was placed in a separatory funnel, and the aqueous layer was extracted with dichloromethane (3×80.00 mL). The organic layers were combined, and the organic layers were washed with a saturated sodium chloride solution (1×150.00 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (dichloromethane: methanol = 400: 1) to obtain compound BA-2 (white solid, yield 70%). 1 HNMR(500MHz, CDCl3)δ9.67(s,1H),4.75(s,1H),4.62(s,1H),3.19-3.16(m,1H),2.86(td,1H,J=11.1,5.8Hz),2.09-1 .99(m,2H),1.92-1.83(m,1H),1.77-1.71(m,3H),1.69(s,3H),1.66-1.59(m,2H),1.56-1.53(m,1H),1.51-1.48(m,1H ),1.47-1.46(m,1H),1.43-1.41(m,4H),1.37-1.35(m,2H),1.33-1.30(m,1H),1.28-1.27(m,1H),1.25-1.23(m,2H),1 .19-1.15(m,1H),1.09-1.02(m,1H),0.96(d,6H,J=5.8Hz),0.91(s,3H),0.81(s,3H),0.75(s,3H),0.68-0.64(m,1H).

[0127] S2. Preparation of compound I-2a: Compound BA-2 (50.00 mg, 0.11 mmol) was placed in a 25 mL two-necked round-bottom flask, 1,2-dichloroethane (8.00 mL) was added to dissolve under nitrogen protection, and cyclohexylmethylamine (43.44 μL, 0.33 mmol) and acetic acid (6.34 μL, 0.11 mmol) were added in sequence. After reacting at 60°C for 4 h, the reaction solution was transferred to an ice bath, methanol (2.00 mL) was added, and sodium cyanoborohydride (20.74 mg, 0.33 mmol) was added, and the reaction was continued at room temperature for 4 h. After the reaction, saturated sodium bicarbonate solution (20.00 mL) was added, the mixed solution was placed in a separatory funnel, the aqueous layer was extracted with dichloromethane (3×20.00 mL), the organic layers were combined, and the organic layers were washed with saturated sodium chloride solution (1×40.00 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain compound Ⅰ-2a (white solid, yield 64%). 1 H NMR(300MHz, CDCl3)δ4.68(s,1H),4.57(s,1H),3.21-3.16(m,1H),2.74-2.70(m,1H) ,2.47-2.38(m,3H),2.18-2.14(m,1H),1.98-1.84(m,3H),1.80-1.74(m,3H),1.70-1. 68(m,7H),1.61-1.57(m,3H),1.54-1.50(m,3H),1.42-1.35(m,6H),1.27-1.17(m,8H ),1.07-1.03(m,5H),0.96(s,6H),0.87-0.83(m,5H),0.76(s,3H),0.70-0.67(m,1H).

[0128] Embodiment 14

[0129] Preparation of compound Ⅰ-2b:

[0130]

[0131] The specific preparation steps are the same as those in Example 13, except that the compound (II) used in S2 is replaced with isopropanolamine to obtain compound I-2b. The detailed preparation method is as described in Example 13. White solid, yield 72%; 1H NMR (500MHz, CDCl3) δ4.66(s,1H),4.56(s,1H),3.80-3.74(m,1H),3.18-3.15(m,1H),2.82-2.68(m,2 H),2.45-2.18(m,3H),1.95-1.84(m,2H),1.80-1.72(m,1H),1.70-1.66(m,4H),1.63-1.57(m,4H),1.5 4-1.49(m,3H),1.42-1.35(m,5H),1.28-1.22(m,3H),1.21-1.17(m,1H),1.14(d,3H,J=6.3Hz),1.07-1 .03(m,2H),1.01(s,3H),0.95(s,6H),0.92-0.85(m,1H),0.81(s,3H),0.75(s,3H),0.70-0.62(m,1H).

[0132] Embodiment 15

[0133] Preparation of compound Ⅰ-2c:

[0134]

[0135] The specific preparation steps are the same as those in Example 13, except that the formula (II) used in S2 is replaced with (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamic acid tert-butyl ester to prepare compound Ⅰ-2c as a white solid with a yield of 75%; 1 H NMR (300MHz, CDCl3) δ5.15 (s, 1H), 4.67 (s, 1H), 4.56 (s, 1H), 3.65-3.58 (m, 6H), 3.53 (t, 2H, J = 5.2Hz), 3.33-3. 27(m,2H),3.20-3.15(m,1H),2.87(t,2H,J=5.3Hz),2.80-2.76(m,1H),2.45-2.36(m,1H),2.29-2.25(m,1H),1. 97-1.71(m,4H),1.67(s,4H),1.63-1.55(m,4H),1.53-1.47(m,2H),1.43-1.41(m,10H),1.38-1.32(m,4H),1.3 0-1.24(m,3H),1.22-1.16(m,2H),1.04-1.02(m,5H),0.95(s,6H),0.81(s,3H),0.75(s,3H),0.68-0.65(m,1H).

[0136] Example 16

[0137] Preparation of compound Ⅰ-2d:

[0138]

[0139] The specific preparation steps are the same as those in Example 13, except that the formula (II) used in S2 is replaced with (3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamic acid tert-butyl ester to prepare compound Ⅰ-2d as a white solid with a yield of 77%; 1 H NMR (300MHz, CDCl3) δ4.98 (s, 1H), 4.68 (s, 1H), 4.60 (s, 1H), 3.72 (t, 2H, J = 5.4Hz), 3.65-3.60 (m, 8H), 3.54 (t, 2H,J=6.0Hz),3.25-3.15(m,5H),3.10-3.06(m,1H),2.66-2.62(m,1H),2.40-2.31(m,1H),2.10-1.98(m,5H),1 .80-1.71(m,3H),1.68(s,4H),1.63-1.58(m,5H),1.43(s,11H),1.40-1.36(m,3H),1.30-1.21(m,5H),1.17-1. 10(m,2H),1.04(s,3H),0.97(d,6H,J=3.1Hz),0.90-0.88(m,1H),0.82(s,3H),0.76(s,3H),0.69-0.66(m,1H).

[0140] Embodiment 17

[0141] Preparation of compound Ⅰ-2e:

[0142]

[0143] The specific preparation steps are the same as those in Example 13, except that the formula (II) used in S2 is replaced with 3,5-difluorobenzylamine to obtain compound Ⅰ-2e as a white solid with a yield of 76%; 1HNMR (500MHz, CDCl3) δ6.90 (d, 2H, J = 7.2Hz), 6.68 (t, 1H, J = 8.9Hz), 4.65 (s, 1H), 4.56 (s, 1H), 3.87-3.73 (m, 2H),3.19-3.16(m,1H),2.65-2.63(m,1H),2.39-2.34(m,1H),2.17-2.15(m,1H),1.93-1.79(m,3H),1.66(s,3 H),1.63-1.57(m,3H),1.53-1.50(m,2H),1.47-1.43(m,2H),1.39-1.31(m,5H),1.25-1.23(m,4H),1.19-1.1 3(m,2H),1.07-1.02(m,2H),0.95(d,6H,J=8.1Hz),0.84(s,3H),0.80(s,3H),0.76(s,3H),0.67-0.63(m,1H).

[0144] Embodiment 18

[0145] Preparation of compound Ⅰ-2f:

[0146]

[0147] The specific preparation steps are the same as those in Example 13, except that the formula (II) used in S2 is replaced by R-(+)-1-phenylethylamine. The prepared compound Ⅰ-2f is a white solid with a yield of 69%. 1 H NMR (300MHz, CDCl3) δ7.38-7.22(m,5H),4.66(s,1H),4.56(s,1H),3.71(q,1H,J=6.6Hz),3.20-3.15( m,1H),2.48-2.33(m,1H),2.15-2.11(m,1H),2.05-1.91(m,2H),1.88-1.79(m,1H),1.67-1.64(m,4H), 1.60-1.55(m,3H),1.48-1.45(m,3H),1.41-1.33(m,7H),1.27-1.22(m,3H),1.18-1.15(m,1H),1.12-1 .02(m,3H),0.97(s,3H),0.91(s,3H),0.88-0.82(m,2H),0.76(s,6H),0.66-0.62(m,1H),0.55(s,3H).

[0148] Embodiment 19

[0149] Preparation of compound Ⅰ-2g:

[0150]

[0151] The specific preparation steps are the same as those in Example 13, except that the formula (II) used in S2 is replaced with 2-furanmethylamine, and the prepared compound I-2 g is a white solid with a yield of 75%. 1 HNMR(300MHz, CDCl3)δ7.36(s,1H),6.33-6.31(m,1H),6.19-6.18(m,1H),4.66(s,1H),4.56(s,1H),3.88-3.72(m,2H ),3.20-3.15(m,1H),2.73-2.69(m,1H),2.42-2.33(m,1H),2.19-2.15(m,1H),1.92-1.77(m,3H),1.72-1.66(m,4H), 1.61-1.55(m,2H),1.52-1.46(m,3H),1.45-1.42(m,1H),1.39-1.32(m,5H),1.28-1.22(m,3H),1.20-1.11(m,2H),1. 04-0.98(m,2H),0.95(d,6H,J=6.6Hz),0.91(s,3H),0.88-0.85(m,1H),0.81(s,3H),0.76(s,3H),0.69-0.60(m,1H).

[0152] Embodiment 20

[0153] Preparation of compound Ⅰ-2h:

[0154]

[0155] The specific preparation steps are the same as those in Example 13, except that the formula (II) used in S2 is replaced with tryptamine to prepare compound Ⅰ-2h as a white solid with a yield of 69%. 1H NMR (300MHz, CDCl3) δ8.01 (s, 1H), 7.65 (d, 1H, J = 7.9Hz), 7.38-7.35 (m, 1H), 7.22-7.09 (m, 2H), 7.05 (s ,1H),4.68(s,1H),4.57(s,1H),3.21-3.16(m,1H),2.99(s,4H),2.77-2.74(m,1H),2.46-2.26(m,2H), 1.91-1.74(m,3H),1.67(s,3H),1.61-1.57(m,2H),1.52-1.43(m,4H),1.38-1.36(m,4H),1.26(s,6H), 1.19-1.13(m,2H),0.99-0.94(m,9H),0.90-0.88(m,2H),0.82(s,3H),0.76(s,3H),0.69-0.66(m,1H).

[0156] Embodiment 21

[0157] Preparation of compound Ⅰ-2i:

[0158]

[0159] The specific preparation steps are the same as those in Example 13, except that the formula (II) used in S2 is replaced with 3,4-methylenedioxybenzylamine to obtain compound Ⅰ-2i as a white solid with a yield of 78%; 1 H NMR(300MHz, CDCl3)δ6.86(s,1H),6.80-6.74(m,2H),5.94-5.93(m,2H),4.65(s,1H),4.56(s,1H),3.81-3.64(m,2H) ,3.20-3.15(m,1H),2.66-2.62(m,1H),2.42-2.33(m,1H),2.18-2.14(m,1H),1.92-1.77(m,3H),1.69-1.64(m,4H),1. 62-1.55(m,3H),1.52-1.48(m,2H),1.44-1.41(m,1H),1.40-1.29(m,5H),1.27-1.18(m,4H),1.17-1.13(m,1H),1.06 -0.99(m,2H),0.96(s,3H),0.93(s,3H),0.91-0.87(m,1H),0.85(s,3H),0.80(s,3H),0.76(s,3H),0.68-0.64(m,1H).

[0160] Application Examples

[0161] The betulinic acid derivative prepared by the present invention is used in treating Alzheimer's disease.

[0162] In this example, the ThT fluorescence experiment was used to determine the effects of the compounds prepared in Examples 1 to 21 on Aβ 42 The degree of inhibition of aggregation reflects its anti-Alzheimer's biological activity. ThT molecules can specifically bind to Aβ 42 The β-sheet structure in the fiber makes it produce strong fluorescence at 480nm under the excitation of 440nm light, but the pure ThT molecule basically does not fluoresce under the excitation of this excitation light. 42 As the fiber aggregation increases, the fluorescence intensity gradually increases. When equilibrium is reached, the fluorescence intensity reaches a plateau and does not continue to increase over time. Therefore, this method can be used to detect Aβ 42 Fiber formation.

[0163] The specific steps for THT fluorescence experimental detection are as follows:

[0164] S1, Aβ 42 Pretreatment of protein:

[0165] 1) The Aβ provided by Hengrui Medicine 42 The powder was dissolved in hexafluoroisopropanol to obtain a solution with a concentration of 1 mg / mL. 42 Dissolve thoroughly.

[0166] 2) After the Aβ 42 The hexafluoroisopropanol solution was ultrasonicated for 10 min to further break up the existing aggregates.

[0167] 3) The Aβ after ultrasonic treatment 42 The hexafluoroisopropanol solution was centrifuged at 16000g for 30 min at 4°C to remove the remaining aggregates.

[0168] 4) From the centrifuged solution, aspirate 75% of the supernatant solution and mix the aspirated supernatant by repeatedly pipetting it.

[0169] 5) Take 300 μL of the mixed solution and dispense it into 1.5 mL EP tubes and freeze at -80°C.

[0170] 6) Place the frozen EP tube in a pre-set freeze dryer and freeze-dry for 24 hours to obtain a white flocculent solid.

[0171] 7) Seal the freeze-dried protein and store it at -20°C for future use.

[0172] S2. Preparation of Aβ 42 fiber:

[0173] 1) Take out the treated Aβ from the -20℃ refrigerator 42 , add 180 μL of 20 mM NaOH solution to dissolve the protein.

[0174] 2) The completely dissolved protein was sonicated for 10 min and centrifuged at 16,000 g for 20 min to remove aggregates.

[0175] 3) Take 75% of the supernatant in a new centrifuge tube, mix well and set aside, use Nanodrop to detect the protein concentration, and prepare Aβ with a concentration of 275 μM based on this. 42 Monomer solution.

[0176] 4) The obtained supernatant protein solution was prepared into Aβ using PBS solution 42 A solution with a final concentration of 0.2 mg / mL was cultured at 37°C, 250 rpm / min for 24 h to obtain Aβ 42 fiber.

[0177] S3. Using the chemicals prepared in Examples 1 to 21 for detection:

[0178] 1) Take out the treated Aβ from the -20℃ refrigerator 42 , use 180 μL of 20 mM NaOH solution to dissolve the protein.

[0179] 2) The completely dissolved protein was sonicated for 10 min and centrifuged at 16,000 g for 20 min to remove aggregates.

[0180] 3) Take 75% of the supernatant in a new centrifuge tube, mix well and set aside to obtain Aβ with a concentration of 275 μM 42 Monomer solution.

[0181] 4) Aβ 42 The monomer and ThT were prepared in a PBS solution at a ratio of 1:1 to a final concentration of 25 μM, and mixed with a certain concentration of the compounds prepared in Examples 1 to 21, respectively, and cultured in a shaking plate instrument at 37°C and 250 rpm / min. Three parallels were set for each group, and the fluorescence value was detected at a fixed point in the experiment.

[0182] In addition, betulinic acid was used to carry out the above THT fluorescence experiment, and an empty bag control group was set up.

[0183] The above multiple groups of test results were processed using Prism 8.0. The specific data are shown in Table 1 below.

[0184] Table 1 Fluorescence intensity ratio

[0185]

[0186]

[0187] like Figure 1 As shown, the ThT fluorescence intensity of the compounds prepared in Examples 1 to 21 was reduced compared with the control group after culturing for 20 hours.

[0188] The above experiments show that after 20 h of culture, the fluorescence intensity increases slowly and reaches a plateau, indicating that Aβ 42 Fibrils and Aβ 42 Monomers and oligomers have reached a dynamic equilibrium in the solution. Adding various compounds has an effect on Aβ 42 The aggregation of all of them was inhibited, and the degree of inhibition was higher than that of the betulinic acid parent. Among them, Example 16 (Compound I-2d) had an inhibitory effect on Aβ 42 The inhibitory effect on aggregation is the most obvious, with the fluorescence intensity accounting for only 16.59%, and it can be further developed as a candidate drug for the treatment of Alzheimer's disease.

[0189] Therefore, the present invention discloses a betulinic acid derivative containing an amide and a secondary amine structure, a preparation method and an application thereof, and structurally modifies betulinic acid to obtain a plurality of betulinic acid derivatives. The in vitro thioflavin (Th) T fluorescence experiment confirms that these compounds can effectively inhibit the expression of β-amyloid protein (Aβ). 42 The aggregation of proteins can be used as candidate drugs for the treatment of Alzheimer's disease and has good application prospects in clinical practice.

[0190] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A betulinic acid derivative containing amide and secondary amine structures, characterized in that: The betulinic acid derivative is one of compound 1 or compound 2, and the structural formula of compound 1 is: The structural formula of compound 2 is: Wherein, R is an alkyl group, a heteroalkyl group, an aryl group or a heteroaryl group.

2. The betulinic acid derivative containing amide and secondary amine structures according to claim 1, characterized in that: In the structural formula of the compound 1, R is one of the following groups:

3. The betulinic acid derivative containing amide and secondary amine structures according to claim 1, characterized in that: In the structural formula of the compound 2, R is one of the following groups:

4. The betulinic acid derivative containing amide and secondary amine structures according to claim 1, characterized in that: The structural formula of the betulinic acid derivative is one of the following compounds I-1a to I-2i:

5. A method for preparing a betulinic acid derivative containing an amide and a secondary amine structure as claimed in any one of claims 1 to 4, characterized in that: The synthetic route of the compound 1 is: The synthetic route of the compound 2 is:

6. The method for preparing a betulinic acid derivative containing amide and secondary amine structures according to claim 5, characterized in that: The specific preparation steps of the compound 1 are: S1. Preparation of compound BA-1: Place betulinic acid in a round-bottom flask, add pyridine to dissolve, then add acetic anhydride, and react at room temperature; adjust the pH of the reaction mixture with 3.00 mol / L hydrochloric acid solution, and stir and mix at room temperature; then place the mixed solution in a separatory funnel, add ethyl acetate to dilute, wash the organic layer with distilled water, then wash with saturated sodium chloride solution, dry the organic layer with anhydrous sodium sulfate, concentrate, and then purify by column chromatography to obtain compound BA-1; S2. Prepare the compound represented by formula (III): place compound BA-1 in two round-bottom flasks, add dichloromethane to dissolve under nitrogen protection, and slowly add oxalyl chloride under ice bath conditions. After reacting for a period of time, directly spin dry to obtain a crude acyl chloride; place the compound represented by formula (II) in a round-bottom flask, add dichloromethane to dissolve, and then add triethylamine to obtain an amine mixed solution; add dichloromethane to the crude acyl chloride obtained above to dissolve it into a solution, add it dropwise to the amine mixed solution under ice bath conditions, and react at room temperature; The reaction mixture was adjusted to pH with 1.00 mol / L hydrochloric acid solution, diluted with dichloromethane, the organic layer was washed with distilled water, and then with saturated sodium chloride solution, the organic layer was dried over anhydrous sodium sulfate, and concentrated to obtain the compound represented by formula (III); S3. Preparation of compound 1: Place the prepared compound of formula (III) in a round-bottom flask, add methanol and tetrahydrofuran to dissolve it, then add sodium hydroxide solution and react at room temperature; after the reaction, use 1.00 mol / L hydrochloric acid solution to adjust the pH of the reaction solution, place the mixed solution in a separatory funnel, add ethyl acetate to extract the aqueous layer, combine the organic layers, wash the organic layers with saturated sodium chloride solution, dry the organic layers with anhydrous sodium sulfate, concentrate and purify by column chromatography to obtain the compound of formula I-1.

7. The betulinic acid derivative containing amide and secondary amine structures according to claim 5, characterized in that: The specific preparation steps of the compound 2 are: S1. Preparation of compound BA-2: Betulin was placed in a 200 mL round-bottom flask, and dichloromethane was added to dissolve it; diacetoxyiodobenzene was added under ice bath conditions, and then TEMPO was added to react at room temperature; after the reaction, a saturated sodium thiosulfate solution was added, and the mixture was stirred and mixed, and then placed in a separatory funnel, and the aqueous layer was extracted with dichloromethane, and the organic layers were combined, and the organic layers were washed with a saturated sodium chloride solution, and the organic layers were dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain compound BA-2; S2. Preparation of compound 2: Compound BA-2 was placed in a two-mouth round-bottom flask, 1,2-dichloroethane was added under nitrogen protection to dissolve, and then the compound represented by formula (II) and acetic acid were added in sequence. After a period of reaction, the reaction solution was moved to an ice bath, methanol was added, and then sodium cyanoborohydride was added, and the reaction was carried out at room temperature for 4 hours. After the reaction was completed, a saturated sodium bicarbonate solution was added, and the mixed solution was placed in a separatory funnel, the aqueous layer was extracted with dichloromethane, the organic layers were combined, the organic layers were washed with a saturated sodium chloride solution, the organic layers were dried with anhydrous sodium sulfate, and the concentrated organic layers were purified by column chromatography to obtain the compound represented by formula I-2.

8. An application of a betulinic acid derivative containing amide and secondary amine structures, characterized in that: The betulinic acid derivative prepared by the preparation method according to any one of claims 5 to 7 is used to treat Alzheimer's disease and inhibit Aβ 42 of gathering.

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