A bumetanide derivative and its preparation method and use

Abstract

The present invention provides a bumetanide derivative, its preparation method, and use, relating to the field of medicinal chemistry. By utilizing the unique properties of the bumetanide skeleton, the organic ligand 4-(2-benzothiazolyl)aniline is modified via an amide reaction to achieve structural modification, thereby obtaining a bumetanide derivative represented by structural formula (I). The bumetanide derivative provided by the present invention has a significant inhibitory effect on abnormal Aβ aggregation and can significantly delay the progression of paralysis in CL4176 nematodes. It has the potential to be developed into a drug for treating Aβ-related diseases such as Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a bumetanide derivative and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disorder that severely impacts the quality of life in the elderly. Its pathological hallmarks include abnormal deposition of β-amyloid (Aβ) plaques, neurofibrillary tangles, and widespread neuronal death. With the increasing global aging population, the incidence of AD has been rising annually, becoming a major public health challenge facing modern society. Currently, treatment for AD primarily focuses on symptom relief, and a cure for AD is still unavailable. Therefore, the development of novel, highly effective AD treatments is of great importance.

[0003] Bumetanide is a diuretic widely used clinically to treat fluid retention caused by heart failure, liver disease, and kidney disease. Studies have shown that bumetanide has some anti-AD effects, improving memory and cognitive function in AD mice. However, as a potent diuretic, high-dose or long-term use of bumetanide can cause serious side effects, such as hypokalemia, hypochloremia, fatigue, muscle aches, and arrhythmias. Its application in anti-AD treatment faces many challenges. Summary of the Invention

[0004] (1) Technical problems solved

[0005] In response to the shortcomings of the existing technology, the present invention structurally modifies bumetanide to obtain a bumetanide derivative with a structural formula as shown in Formula (I). This compound can effectively inhibit the aggregation of β-amyloid protein and significantly delay the paralysis process of CL4176 nematodes. It has the prospect of being developed into a drug for treating β-amyloid protein-related diseases.

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

[0007] (2) Technical solution

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

[0009] In the first aspect, the present invention provides a compound, which is a derivative of bumetanide, and its chemical structure is shown in (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 a 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 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 comprising an effective amount of formula (I) and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is in the form of 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), comprising the following steps: mixing bumetanide and 4-(2-benzothiazolyl)aniline in a molar ratio of 1:1-2 and dissolving the mixture in dichloromethane; then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole in a molar ratio of 1:1; and stirring at room temperature for more than 24 hours. After the reaction is completed, dichloromethane is removed, and ethyl acetate is added as the organic phase for extraction. The organic phase is washed with a saturated sodium bicarbonate solution for more than three times, and then the organic phase is separated. The organic phase is concentrated and dried, and then separated by column to obtain the compound represented by formula (I).

[0016] (3) Beneficial effects

[0017] The present invention utilizes the unique properties of the bumetanide skeleton to modify the organic ligand 4-(2-benzothiazolyl)aniline via an amide reaction, achieving structural modification and yielding a bumetanide derivative represented by structural formula (I). The bumetanide derivative provided by the present invention exhibits significant inhibitory effects on abnormal Aβ aggregation and can significantly slow the progression of paralysis in CL4176 nematodes. It holds promise for development as a drug for Aβ-related diseases, such as Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Different drugs for Cu 2+ Induction of Aβ 25-35 Aggregation inhibition results: *P<0.05, **P<0.01, ***P<0.001 compared with curcumin.

[0019] Figure 2 The paralysis rates of the CL4176 nematode paralysis model within 36 h are shown. Compared with the control group, *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with 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. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0021] Example 1

[0022] Preparation of compound Ⅰ:

[0023]

[0024] Bumetanide (compound 1, 0.5 mmol) and 4-(2-benzothiazolyl)aniline (0.5 mmol) were dissolved in dichloromethane, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.6 mmol) and 1-hydroxybenzotriazole (HOBT, 0.6 mmol) were added. The mixture was stirred at room temperature for 24 hours. After the reaction, dichloromethane was removed by distillation under reduced pressure, and then ethyl acetate was added. The organic phase was washed 2-3 times with saturated sodium bicarbonate solution. The organic phase was concentrated and dried, and then separated by column to obtain the compound represented by formula (I).

[0025] Spectral data of the compound represented by formula (I): 1 H NMR (500MHz, DMSO-d6) δ10.67(s,1H),8.13(m,3H),8.04(d,J=8.1Hz,1H),8.01(m,2H),7.71(m,1H),7.55(m,1H),7.47(m,2H),7.32-7.27(m, 4H),7.03(t,J=7.3Hz,1H),6.89(d,J=8.1Hz,2H),5.01(t,J=5.6Hz,1H),3.15-3.11(m,2H),1.40(m,2H),1.13(m,2H),0.78(t,J=7.4Hz,3H).

[0026] Experimental Example 1

[0027] 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

[0028] 1 Experimental Materials

[0029] 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 Yien Chemical Technology Co., Ltd.

[0030] 2 Experimental methods

[0031] 2.1 Solution preparation

[0032] HEPES buffer: Weigh an appropriate amount of HEPES and dissolve it in ultrapure water to prepare a 10 mM buffer (pH 7.2);

[0033] Thioflavin T (ThT) solution: Weigh an appropriate amount of thioflavin T (ThT) and dissolve it in HEPES buffer to prepare an 80 μM solution;

[0034] Cu 2+ Solution: Weigh an appropriate amount of CuCl2 and dissolve it in HEPES buffer to prepare a 200 μM solution;

[0035] 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;

[0036] Curcumin solution: Weigh an appropriate amount of curcumin and dissolve it in DMSO to prepare a 200 μM solution;

[0037] Target compound solution: Bumetanide (target compound 1), a 1:1 molar ratio mixture of bumetanide and 4-(2-benzothiazolyl)aniline (target compound 2), and the compound represented by formula (I) (target compound 3) were weighed and dissolved in DMSO to prepare a 200 μM solution.

[0038] 2.2 The cells were processed in the following groups, with 3 replicates per group:

[0039] Experimental group: 166 μL HEPES buffer, 10 μL Cu 2+ solution, 4 μL Aβ 25-35 solution, 10 μL target compound solution.

[0040] Curcumin group (positive drug group): 166 μL HEPES buffer, 10 μL Cu 2+ solution, 4 μL Aβ 25-35 solution, 10 μL curcumin solution.

[0041] Blank group: 170 μL HEPES buffer, 10 μL Cu 2+ solution, and 10 μL of compound solution corresponding to the experimental group or curcumin group.

[0042] Control group: 166 μL HEPES buffer, 10 μL Cu 2+ solution, 4 μL Aβ 25-35 solution, 10 μL DMSO solution.

[0043] Solvent group: 170 μL HEPES buffer, 10 μL Cu 2+ solution, 10 μL DMSO solution.

[0044] Add them to black 96-well plates and incubate them at 37°C for 24 hours. After incubation, 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. 2+ Induction of Aβ 25-35 The aggregation inhibition percentage is calculated as follows: inhibition percentage = 100-(IF i -IF0) / (IF c -IF r )×100. i : Fluorescence value of the experimental group, IF0: Fluorescence value of the blank group, IF c : Fluorescence value of the control group, IF r : Fluorescence value of the solvent group.

[0045] 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 inhibitory effect of bumetanide derivatives of formula (I) prepared by the present invention on Cu 2+ Induction of Aβ 25-35 The inhibitory effect on Aβ aggregation was significantly greater than that of bumetanide and a mixture of bumetanide and 4-(2-benzothiazolyl)aniline. The above results demonstrate 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 superior to that of bumetanide and a mixture of bumetanide and 4-(2-benzothiazolyl)aniline.

[0046] Experimental Example 2

[0047] Study on the effect of the bumetanide derivatives represented by formula (I) provided by the present invention on the CL4176 nematode paralysis model

[0048] 1 Experimental Materials

[0049] MgSO4 was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., agar powder, yeast powder, cholesterol, tryptone, NaCl, and CaCl2 were all purchased from Beijing Solebeau Technology Co., Ltd., Na2HPO4·12H2O and KH2PO4 were purchased from Sinopharm Chemical Reagent Co., Ltd., and 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 were purchased from the Caenorhabditis elegans Genetics Center in the United States.

[0050] 2. Solution Preparation

[0051] 2.1M9 buffer preparation

[0052] 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 before use.

[0053] 2.2 Preparation of NGM culture plates

[0054] Weigh 3.0g sodium chloride, 2.5g protein chen, and 17g agar into a conical flask. Add 975mL of deionized water and autoclave. When the sterilization solution temperature drops to approximately 55°C, add 0.5mL of 1M calcium chloride solution, 1mL of 5mg / mL cholesterol solution, 1mL of 1M magnesium sulfate solution, and 25mL of potassium phosphate buffer. Use a pipette to add the prepared culture medium to the corresponding petri dishes or well plates and cool overnight to solidify.

[0055] 3 Experimental methods

[0056] 3.1 Evenly spread 200 μL of each target compound 1-3 solution (prepared as described in Section 2.1 of Experimental Example 1) onto a freshly prepared NGM plate and allow to dry naturally at room temperature. Add 50 μL of OP50 bacterial solution to the center of the plate and allow to dry overnight. Synchronized C. elegans of the relevant strains were inoculated onto the drug-containing plates. A separate control group was treated with a 0.1% DMSO-M9 solution.

[0057] 3.2 The synchronized CL4176 strain of Caenorhabditis elegans was cultured at 16°C for 48 hours, ie, when the Caenorhabditis elegans reached the L3 stage, the culture plate was transferred to 25°C to induce transcriptional expression of the Aβ gene.

[0058] 3.3 Transfer the culture plate to 25°C and count the number of paralyzed C. elegans at 36 hours. Paralyzed C. elegans exhibit partial rigidity of the trunk, resulting in motor impairment or even loss of motor function, and the head may twist.

[0059] 4 Detection indicators

[0060] For paralysis rate testing, each group should have at least 30 nematodes. Paralysis rate within 36 hours (%) = paralyzed nematode count / total nematode count × 100%. Each experiment was repeated three times.

[0061] 5 Results

[0062] The results are as follows Figure 2 As shown, the bumetanide derivatives of formula (I) showed a significantly lower paralysis rate within 36 hours than the control group (P < 0.05). Furthermore, the bumetanide derivatives of formula (I) showed a significantly lower paralysis rate within 36 hours than bumetanide and a mixture of bumetanide and 4-(2-benzothiazolyl)aniline. These results demonstrate that the bumetanide derivatives of formula (I) provided herein can significantly delay paralysis in CL4176 nematodes, with significantly better efficacy than bumetanide and a mixture of bumetanide and 4-(2-benzothiazolyl)aniline.

[0063] The above results indicate that the bumetanide derivatives of formula (I) provided by the present invention can inhibit Aβ aggregation and 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.

[0064] 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 they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. 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 various embodiments of the present invention.

Claims

1. Use of a bumetanide derivative represented by the chemical formula (I) in the preparation of a drug for inhibiting abnormal aggregation of β-amyloid protein;

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