Bumetanib derivative and preparation and application thereof

Abstract

The invention provides a bumetanib derivative as well as preparation and application thereof, and relates to the field of medicinal chemistry. According to the invention, bumetanib is taken as a basic structural framework, D-pinitol is taken as an organic ligand for structural modification, and the bumetanib derivative with the structural formula (I) is finally obtained. The bumetanib derivative provided by the invention can effectively inhibit abnormal aggregation of beta-amyloid protein and remarkably delay the CL4176 nematode paralysis process, and has the prospect of being developed into a medicine for resisting beta-amyloid protein related diseases such as Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to bumetanide derivatives and preparation and application thereof. Background Art

[0002] Alzheimer's Disease (AD) is a neurodegenerative disease that seriously affects the quality of life of the elderly. Its pathological characteristics mainly include plaques formed by abnormal deposition of β-amyloid (Aβ), neurofibrillary tangles, and extensive neuronal death. With the intensification of the global aging trend, the incidence of AD has increased year by year, and it has become one of the major public health challenges facing modern society. At present, the treatment of AD mainly focuses on symptom relief, and there is no drug that can cure AD. Therefore, it is of great significance to develop new and efficient AD therapeutic drugs.

[0003] Bumetanide is a diuretic that is mainly used to treat fluid retention caused by heart failure, liver disease, and kidney disease, and is widely used in clinical practice. Studies have shown that bumetanide has a certain anti-AD effect and can improve the memory and cognitive function of AD mice. However, as a powerful diuretic, bumetanide can cause serious side effects when used in large doses or for a long time, such as hypokalemia, hypochloremia, fatigue, muscle soreness, arrhythmia, etc., and its application in anti-AD treatment still faces many challenges. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies of the prior art, the present invention structurally transforms bumetanide to obtain a bumetanide derivative having a structural formula as shown in formula (I). The compound can effectively inhibit the abnormal aggregation of β-amyloid protein and significantly delay the paralysis process of CL4176 nematodes, and has the prospect of being developed into a drug against β-amyloid protein-related diseases.

[0006] Provided are a compound for treating or preventing Alzheimer's disease and a preparation method thereof.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] In a first aspect, the present invention provides a bumetanide derivative having a chemical structural formula (I):

[0010]

[0011] In a second aspect, the present invention provides a compound as represented by formula (I) or a pharmaceutically acceptable salt or solvent compound thereof.

[0012] In a third aspect, the present invention provides the use of the compound represented by formula (I) in the preparation of a drug for inhibiting abnormal aggregation of β-amyloid protein.

[0013] In a fourth aspect, the present invention provides the use of the compound represented by formula (I) in the preparation of a drug for treating and / or preventing Alzheimer's disease.

[0014] In a fifth aspect, the present invention provides a pharmaceutical composition, which comprises an effective amount of formula (I) and a pharmaceutically acceptable carrier. The pharmaceutical composition is a capsule, tablet, injection, granule, pill or powder.

[0015] In a sixth aspect, the present invention provides a method for preparing the compound represented by formula (I), the specific steps of which are:

[0016] (1) 1-5 mmol D-pine alcohol and 1-5 mL 2,2-dimethoxypropane are dissolved in 5-25 mL acetone, 0.04-0.2 mmol p-toluenesulfonic acid is added at room temperature and stirred overnight; after the reaction is completed, the solvent is concentrated under reduced pressure, and then a saturated sodium bicarbonate solution is added, and then ethyl acetate is used as an organic phase for extraction; the organic phase is washed with deionized water for more than 3 times, the organic phase is separated, the organic phase is dried with anhydrous sodium sulfate overnight, and then separated by column chromatography to obtain compound 1; the structural formula of compound 1 is shown in formula (II)

[0017]

[0018] (2) mixing bumetanide and compound 1 at a molar ratio of 1:1-2 and dissolving in dichloromethane to obtain a mixture 1;

[0019] (3) Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine in a molar ratio of 1:1 were added to the mixture 1, and the mixture was stirred at room temperature for more than 24 hours. After the reaction, dichloromethane was removed, and then ethyl acetate was added as an organic phase for extraction. The organic phase was washed with a saturated sodium bicarbonate solution for more than 3 times, and then the organic phase was separated, and the organic phase was concentrated and dried to obtain a crude product;

[0020] (4) The crude product is then dissolved in ethanol, and 2-4 mmol of concentrated hydrochloric acid is added, and the mixture is stirred at room temperature for more than 12 hours. After the reaction is completed, ethyl acetate is added to the reaction solution as an organic phase for extraction, and the mixture is washed with deionized water for more than 3 times, and then washed with a saturated sodium bicarbonate solution for more than 3 times. The organic phase is then separated, concentrated and dried, and then separated by a column to obtain the compound represented by formula (I).

[0021] (III) Beneficial effects

[0022] The present invention uses bumetanide as a basic structural skeleton and D-pinitol as an organic ligand to modify the structure thereof, and finally obtains a bumetanide derivative shown in the structural formula (I). The bumetanide derivative provided by the present invention can effectively inhibit the abnormal aggregation of β-amyloid protein, significantly delay the paralysis process of CL4176 nematodes, and has the prospect of being developed into a drug for anti-β-amyloid protein related diseases such as Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Different drugs for Cu 2+ Induction of Aβ 25-35 Aggregation inhibition results: *P<0.05, ***P<0.001 compared with curcumin.

[0024] Figure 2 The paralysis rate of the CL4176 nematode paralysis model within 36 hours, among which, compared with the control group, *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] Compound I was prepared as follows

[0028]

[0029] D-pine alcohol (1 mmol) and 2,2-dimethoxypropane (1 mL) were dissolved in acetone (5 mL), p-toluenesulfonic acid (0.04 mmol) was added at room temperature and stirred overnight. After the reaction was completed, a saturated sodium bicarbonate solution was added, the solvent was concentrated under reduced pressure, and then ethyl acetate was used as the organic phase for extraction; the organic phase was washed with deionized water for more than 3 times, the organic phase was separated, and the organic phase was dried with anhydrous sodium sulfate, and then the organic phase was separated by column chromatography to obtain compound 1.

[0030] Bumetanide (0.5 mmol) and compound 1 (0.5 mmol) were dissolved in dichloromethane, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.6 mmol) and 4-dimethylaminopyridine (DMAP, 0.6 mmol) were added, and stirred at room temperature for 24 hours. After the reaction was completed, dichloromethane was removed by distillation under reduced pressure, and then ethyl acetate was added. The organic phase was washed 2-3 times with a saturated sodium bicarbonate solution. After the organic phase was concentrated and dried, the crude product was directly used in the next step. The crude product of the previous step was dissolved in ethanol, concentrated hydrochloric acid (2 mmol) was added, and stirred at room temperature for 12 hours. Ethyl acetate was added to the reaction solution, washed three times with water, and then washed 2-3 times with a saturated sodium bicarbonate solution. The organic phase was concentrated and dried, and then column chromatography was performed to obtain the compound represented by formula (I).

[0031] Spectral data of compound 1: 1 H NMR(500MHz,Chloroform-d)δ4.25-4.19(m,4H),3.61(m,1H),3.59(s,3H),3.19(m,1H),2.84(s,1H),1.52(s,6H),1.35(s,6H).

[0032] Spectral data of the compound represented by formula (Ⅰ): 1 H NMR (500 MHz, DMSO-d 6 )δ7.75(d,J=2.0Hz,1H),7.47(d,J=2.0Hz,1H),7.37(s,2H),7.28(m,2H),7.02(t ,J=7.3Hz,1H),6.86(m,2H),5.22(t,J=9.8Hz,1H),5.12(t,J=5.8Hz,1H),5.06(m, 2H),4.81(d,J=6.4Hz,1H),4.73(d,J=6.9Hz,1H),3.82(m,1H),3.74(m,3H),3.42( m,1H),3.36(s,3H),3.09(m,2H),1.39(m,2H),1.13(m,2H),0.79(t,J=7.3Hz,3H).

[0033] Experimental Example 1

[0034] The bumetanide derivatives shown in formula (I) provided by the present invention have an effect on Cu 2+ Induction of Aβ 25-35 Exploring the impact of aggregation

[0035] 1 Experimental Materials

[0036] Aβ 25-35purchased from Beijing Bioson Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), thioflavin T (ThT), and curcumin were all purchased from Shanghai En Chemical Technology Co., Ltd.

[0037] 2 Experimental methods

[0038] 2.1 Solution preparation

[0039] HEPES buffer: Weigh an appropriate amount of HEPES and dissolve it in ultrapure water to prepare a 10 mM buffer (pH 7.2);

[0040] Thioflavin T (ThT) solution: Weigh an appropriate amount of thioflavin T (ThT) and dissolve it in HEPES buffer to prepare an 80 μM solution;

[0041] Cu 2+ Solution: Weigh an appropriate amount of CuCl 2 , dissolved in HEPES buffer to prepare a 200 μM solution;

[0042] Aβ 25-35 Solution: Weigh an appropriate amount of Aβ 25-35 Dissolved in HEPES buffer (containing 37.5% DMSO) to prepare a 500 μM solution;

[0043] Curcumin solution: weigh an appropriate amount of curcumin and dissolve it in DMSO to prepare a 200 μM solution;

[0044] Target compound solution: Bumetanide (target compound 1), a mixture of bumetanide and D-pinitol in a molar ratio of 1:1 (target compound 2), and the compound represented by formula (I) were weighed and dissolved in DMSO to prepare a 200 μM solution.

[0045] 2.2 Treat the cells in the following groups, with 3 replicates per group:

[0046] Experimental group: 166 μL HEPES buffer, 10 μL Cu 2+ Solution, 4 μL Aβ 25-35 solution, 10 μL target compound solution.

[0047] Curcumin group (positive drug group): 166 μL HEPES buffer, 10 μL Cu 2+ Solution, 4 μL Aβ 25-35 solution, 10 μL curcumin solution.

[0048] Blank group: 170 μL HEPES buffer, 10 μL Cu 2+ solution, 10 μL of compound solution corresponding to the experimental group or curcumin group.

[0049] Control group: 166 μL HEPES buffer, 10 μL Cu 2+ Solution, 4 μL Aβ 25-35 solution, 10 μL DMSO solution.

[0050] Solvent group: 170 μL HEPES buffer, 10 μL Cu 2+ solution, 10 μL DMSO solution.

[0051] Add them to black 96-well plates and incubate them at 37°C for 24 hours. After the incubation is completed, add 10 μL of ThT solution to each group and incubate them at 37°C for 3 hours. Use a multifunctional microplate reader to measure the fluorescence value at an excitation wavelength of 440 nm and an emission wavelength of 480 nm. Calculate the target compound and positive drug for Cu 2+ Induction of Aβ 25-35 The aggregation inhibition percentage is calculated as follows: inhibition percentage = 100-(IF i -IF 0 ) / (IF c -IF r )×100. i : Fluorescence value of the experimental group, IF 0 : Fluorescence value of blank group, IF c : Fluorescence value of the control group, IF r : Fluorescence value of the solvent group.

[0052] The results are as follows Figure 1 As shown, the bumetanide derivatives of formula (I) prepared by the present invention have an effect on Cu 2+ Induction of Aβ 25-35 The inhibition percentage of aggregation of the bumetanide derivatives of formula (I) prepared by the present invention was significantly higher than that of curcumin, and the difference was extremely significant (P<0.01). 2+ Induction of Aβ 25-35 The inhibition percentage of aggregation is significantly higher than that of bumetanide and a mixture of bumetanide and D-pinitol. The above results show that the bumetanide derivative represented by formula (I) provided by the present invention has a significant inhibitory effect on Aβ aggregation, and the effect is significantly better than that of bumetanide and a mixture of bumetanide and D-pinitol.

[0053] Experimental Example 2

[0054] Study on the effect of the bumetanide derivatives represented by formula (I) provided by the present invention on the CL4176 nematode paralysis model

[0055] 1 Experimental Materials

[0056] MgSO 4Agar powder, yeast powder, cholesterol, tryptone, NaCl, CaCl were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. 2 All were purchased from Beijing Solebow Technology Co., Ltd. 2 HPO 4 12H 2 O.KH 2 PO 4 All were purchased from Sinopharm Chemical Reagent Co., Ltd. The transgenic AD model Caenorhabditis elegans strains CL802 (smg-1(cc546)I; 109rol-6(su1006)II), CL4176 [dvIs27[myo-3p::A-Beta(1-42)::let-851 3'UTR)+rol-6(su1006)]X] and uracil-deficient Escherichia coli OP50 (Escherichia Coli OP50) were purchased from the Caenorhabditis elegans Genetics Center in the United States.

[0057] 2. Solution Preparation

[0058] 2.1M9 buffer preparation

[0059] Weigh 0.6 g of sodium hydrogen phosphate, 0.3 g of potassium dihydrogen phosphate, 0.5 g of sodium chloride, and 0.025 g of magnesium sulfate (dehydrated), dissolve them in 100 mL of deionized water, and sterilize them at high temperature for later use.

[0060] 2.2 Preparation of NGM culture plates

[0061] Weigh 3.0g sodium chloride, 2.5g protein Chen, and 17g agar into a conical flask, add 975mL deionized water, and sterilize them by high pressure. When the temperature of the sterilized solution drops to about 55°C, add 0.5mL 1M calcium chloride solution, 1mL 5mg / mL cholesterol solution, 1mL 1M magnesium sulfate solution, and 25mL potassium phosphate buffer in sequence. Use a pipette to add the prepared culture medium liquid to the corresponding plate or well plate, and cool and solidify overnight.

[0062] 3 Experimental methods

[0063] 3.1 Take 200 μL of target compound 1-3 solution (preparation method is the same as that of Experimental Example 1, Section 2.1) and evenly spread it on the freshly prepared NGM plate and dry it naturally at room temperature. Add 50 μL of OP50 bacterial solution to the center of the plate and dry it overnight. Inoculate the synchronized Caenorhabditis elegans of the relevant strains into the drug-containing plate, and set up a control group and treat it with 0.1% DMSO-M9 solution.

[0064] 3.2 The synchronized CL4176 strain of Caenorhabditis elegans was cultured at 16°C for 48 hours, ie, when the Caenorhabditis elegans grew to the L3 stage, the culture plate was transferred to 25°C to induce the transcriptional expression of the Aβ gene.

[0065] 3.3 The culture plate was transferred to 25°C culture condition, and the number of paralyzed C. elegans was recorded at 36 hours. The paralyzed C. elegans individuals showed partial rigidity of the trunk, resulting in movement disorders or even loss of movement function, and the head could be twisted.

[0066] 4. Detection indicators

[0067] In the paralysis rate detection experiment, the number of nematodes in each group is not less than 30. The paralysis rate of nematodes within 36 hours (%) = paralyzed nematode count / total number of nematodes × 100%. Each group of experiments was repeated 3 times.

[0068] 5 Results

[0069] The results are as follows Figure 2 As shown, the paralysis rate of nematodes within 36 hours of the bumetanide derivatives shown in formula (Ⅰ) was significantly lower than that of the control group, and the difference was significant (P<0.05); at the same time, the paralysis rate of nematodes within 36 hours of the bumetanide derivatives shown in formula (Ⅰ) was significantly lower than that of bumetanide and the mixture of bumetanide and D-pinitol. The above results show that the bumetanide derivatives shown in formula (Ⅰ) provided by the present invention can significantly delay the paralysis time of CL4176 nematodes, and the effect is significantly better than that of bumetanide and the mixture of bumetanide and D-pinitol.

[0070] The above results indicate that the bumetanide derivatives of formula (I) provided by the present invention can inhibit Aβ aggregation, delay the paralysis process of CL4176 nematodes, and have the prospect of being developed into drugs against Aβ-related diseases such as Alzheimer's disease.

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A bumetanide derivative, characterized in that The chemical structural formula of the bumetanide derivative is shown in (I) 2. A compound represented by formula (I) or a pharmaceutically acceptable salt or solvent compound thereof.

3. Use of the compound represented by formula (I) in the preparation of a drug for inhibiting abnormal aggregation of β-amyloid protein.

4. Use of the compound represented by formula (I) in the preparation of a drug for treating and / or preventing Alzheimer's disease.

5. A pharmaceutical composition, characterized in that The pharmaceutical combination comprises an effective amount of formula (I) and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition according to claim 4, characterized in that: The pharmaceutical composition is in the form of capsules, tablets, injections, granules, pills or powders.

7. The method for preparing a compound according to claim 1, characterized in that: The compound represented by formula (I) is prepared according to the following steps: (1) 1-5 mmol D-pine alcohol and 1-5 mL 2,2-dimethoxypropane are dissolved in 5-25 mL acetone, 0.04-0.2 mmol p-toluenesulfonic acid is added at room temperature and stirred overnight; after the reaction is completed, the solvent is concentrated under reduced pressure, and then a saturated sodium bicarbonate solution is added, and then ethyl acetate is used as an organic phase for extraction; the organic phase is washed with deionized water for more than 3 times, the organic phase is separated, the organic phase is dried with anhydrous sodium sulfate overnight, and then separated by column chromatography to obtain compound 1; the structural formula of compound 1 is shown in formula (II) (2) mixing bumetanide and compound 1 at a molar ratio of 1:1-2 and dissolving in dichloromethane to obtain a mixture 1; (3) Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine in a molar ratio of 1:1 were added to the mixture 1, and the mixture was stirred at room temperature for more than 24 hours. After the reaction, dichloromethane was removed, and then ethyl acetate was added as an organic phase for extraction. The organic phase was washed with a saturated sodium bicarbonate solution for more than 3 times, and then the organic phase was separated, and the organic phase was concentrated and dried to obtain a crude product; (4) The crude product is then dissolved in ethanol, and 2-4 mmol of concentrated hydrochloric acid is added, and the mixture is stirred at room temperature for more than 12 hours. After the reaction is completed, ethyl acetate is added to the reaction solution as an organic phase for extraction, and the mixture is washed with deionized water for more than 3 times, and then washed with a saturated sodium bicarbonate solution for more than 3 times. The organic phase is then separated, concentrated and dried, and then separated by a column to obtain the compound represented by formula (I).

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