Compound, preparation method and use in treating Alzheimer's disease

Abstract

The present invention discloses a compound, a preparation method and a use for treating Alzheimer's disease. The compound has a novel structure and a simple synthesis method and is easy to produce. Anti-Aβ aggregation experiments show that the compound has anti-Aβ aggregation activity that is stronger than that of positive compounds. Those skilled in the art know that Aβ aggregation refers to the process in which β-amyloid protein spontaneously forms aggregates in the brain, and abnormal aggregation of Aβ protein is one of the main pathological characteristics of AD. The present invention further uses AD model nematodes as a model organism to prove that the compound has a significant effect of inhibiting paralysis in AD model nematodes. Therefore, the compound provided by the present invention has the prospect of being developed into a drug for treating diseases caused by abnormal aggregation of β-amyloid protein, such as AD.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry and relates to the discovery, preparation and use of new compounds, and specifically to a compound, a preparation method and use for treating Alzheimer's disease. 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 steadily increased, posing a significant public health challenge for modern society.

[0003] Currently, AD treatments primarily focus on symptom relief, and there is no cure for AD. Therefore, developing novel, highly effective AD treatments is of great significance. Summary of the Invention

[0004] The first purpose of the present invention is to provide a compound with a novel structure, the second purpose is to provide a method for preparing the compound, and the third purpose is to provide medical uses of the compound.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] A compound having a structural formula as shown in Formula I or II, or a pharmaceutically acceptable salt or solvate thereof;

[0007]

[0008] A method for preparing the compound represented by the above formula I, wherein the raw materials include (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid and 4-(2-benzothiazolyl)aniline.

[0009] Preferably, the molar ratio of (2S)-2-(2-oxopyrrolidin-1-yl)butanoic acid and 4-(2-benzothiazolyl)aniline is 1:1.

[0010] A method for preparing the compound represented by the above formula II, wherein the raw materials include (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid and riluzole.

[0011] Preferably, the molar ratio of (2S)-2-(2-oxopyrrolidin-1-yl)butanoic acid to riluzole is 1:1.

[0012] A use of the compound represented by the above formula I or II or a pharmaceutically acceptable salt or solvate thereof for preparing a drug for treating diseases caused by abnormal aggregation of β-amyloid protein.

[0013] Preferably, the disease caused by abnormal aggregation of β-amyloid protein is Alzheimer's disease.

[0014] A pharmaceutical preparation, comprising the compound represented by the above formula I or II or a pharmaceutically acceptable salt or solvate thereof as an active ingredient, and prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier or excipient.

[0015] The pharmaceutical preparation is used for preparing medicines for treating diseases caused by abnormal aggregation of β-amyloid protein.

[0016] Preferably, the disease caused by abnormal aggregation of β-amyloid protein is Alzheimer's disease.

[0017] Beneficial effects:

[0018] 1. The present invention provides two novel compounds, the synthesis methods of these two new compounds are simple and easy to produce.

[0019] 2. Anti-Aβ aggregation experiments show that the compounds provided by the present invention have stronger anti-Aβ aggregation activity than the positive compounds. Those skilled in the art know that Aβ aggregation refers to the process in which β-amyloid protein spontaneously forms aggregates in the brain, and abnormal aggregation of Aβ protein is one of the main pathological characteristics of AD. Aβ protein spontaneously forms aggregates such as oligomers, fibrils and fibers in the brain. These aggregates can cause dysfunction and death of nerve cells, and eventually lead to dementia. The present invention further uses AD model nematodes as a model organism to prove that these two new compounds have a significant effect of inhibiting paralysis in AD model nematodes. Therefore, the compounds provided by the present invention have the prospect of being developed into drugs for treating diseases caused by abnormal aggregation of β-amyloid protein, such as AD. DETAILED DESCRIPTION

[0020] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0021] Example 1: Preparation of Compound (I)

[0022]

[0023] (2S)-2-(2-Oxopyrrolidin-1-yl)butyric acid (0.5 mmol) and 4-(2-benzothiazolyl)aniline (0.5 mmol) were dissolved in 10 mL of dichloromethane, and 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 h. After the reaction, dichloromethane was removed by distillation under reduced pressure. Subsequently, 10 mL of ethyl acetate was added, stirred, and washed three times with 5 mL of saturated sodium bicarbonate solution. The ethyl acetate solution was concentrated and loaded onto normal phase silica gel column chromatography. The solution was isocratically eluted with a mixed solvent of petroleum ether / ethyl acetate in a volume ratio of 2:1. The solution was detected by TLC. The eluate corresponding to the target compound was collected, concentrated, and dried to obtain the compound of formula (I) with an HPLC purity of 95.9%. 1 HNMR(500MHz,DMSO-d6)δ8.96(s,1H),8.03(m,3H),7.89(m,1H),7.70–7.68(m,2H),7.49–7.35(m,2H), 4.59(t,J=7.8Hz,1H),3.51(m,2H),2.50(m,2H),2.12–2.04(m,3H),1.85(m,1H),0.98(t,J=7.4Hz,3H).

[0024] Example 2: Preparation of compound (II)

[0025]

[0026] (2S)-2-(2-Oxopyrrolidin-1-yl)butyric acid (0.5 mmol) and riluzole (0.5 mmol) were dissolved in 10 mL of dichloromethane, and 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 h. After the reaction, the dichloromethane was removed by distillation under reduced pressure. Then, 10 mL of ethyl acetate was added, stirred, and washed three times with 5 mL of saturated sodium bicarbonate solution. The ethyl acetate solution was concentrated and loaded onto a normal phase silica gel column chromatography. The solution was isocratically eluted with a mixed solvent of petroleum ether / ethyl acetate in a volume ratio of 2:1. The solution was detected by TLC. The eluate corresponding to the target compound was collected, concentrated, and dried to obtain the compound of formula (II) with an HPLC purity of 97.8%. 1H NMR(500MHz,DMSO-d6)δ11.38(br,1H),7.73(m,1H),7.63(m,1H),7.25(m,1H),4.91(m,1H),3. 64(m,1H),3.56(m,1H),2.59(t,J=7.9Hz,2H),2.10(m,3H),1.89(m,1H),0.97(t,J=7.4Hz,3H).

[0027] Example 3: Compounds (I) and (II) on Cu 2+ Induction of Aβ 25-35 The impact of aggregation

[0028] 1. Experimental Materials

[0029] Aβ 25-35 purchased from Beijing Bioson Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and thioflavin T (ThT) were purchased from Shanghai Yien Chemical Technology Co., Ltd. Compounds (I) and (II) were prepared according to Examples 1 and 2, respectively. Homemade ultrapure water was used in the experiments.

[0030] 2. Experimental Methods

[0031] 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 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] Sample stock solution to be tested:

[0037] (1) Positive compound stock solution: Weigh an appropriate amount of curcumin and dissolve it in DMSO to prepare a 200 μM solution;

[0038] (2) Compound (I) stock solution: Weigh an appropriate amount of compound (I) and dissolve it in DMSO to prepare a 200 μM solution;

[0039] (3) Compound (II) stock solution: Weigh an appropriate amount of compound (II) and dissolve it in DMSO to prepare a 200 μM solution;

[0040] (4) Comparative sample 1 mother solution: Weigh appropriate amounts of (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid and 4-(2-benzothiazolyl)aniline and dissolve them in DMSO to prepare a mixed solution of 200 μM (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid + 200 μM 4-(2-benzothiazolyl)aniline;

[0041] (5) Comparative sample 2 mother solution: Weigh appropriate amounts of (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid and riluzole and dissolve them in DMSO to prepare a mixed solution of 200 μM (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid + 200 μM riluzole.

[0042] 2. Grouping, Modeling, and Drug Administration

[0043] Experimental group: 166 μL HEPES buffer, 10 μL Cu 2+ Solution, 4 μL Aβ 25-35 solution, 10 μL of the mother solution of the sample to be tested.

[0044] Blank group: 170 μL HEPES buffer, 10 μL Cu 2+ solution, 10 μL of the mother solution of the sample to be tested.

[0045] Control group: 166 μL HEPES buffer, 10 μL Cu 2+ Solution, 4 μL Aβ 25-35 solution, 10 μL DMSO solution.

[0046] Solvent group: 170 μL HEPES buffer, 10 μL Cu 2+ solution, 10 μL DMSO solution.

[0047] Add to black 96-well plates, with 3 replicates per group. Incubate at 37°C for 24 hours. After incubation, add 10 μL of ThT solution to each group and incubate at 37°C for 3 hours. Measure the fluorescence value using a multifunctional microplate reader at an excitation wavelength of 440 nm and an emission wavelength of 480 nm. Calculate the Cu ions of the test sample according to the following formula. 2+ Induction of Aβ 25-35 Percent inhibition of aggregation.

[0048] Inhibition percentage (%) = [1-(IF i -IF0) / (IF c -IF r )]×100%.

[0049] IF in the formulai represents the fluorescence value of the experimental group, IF0 represents the fluorescence value of the blank group, and IF c represents the fluorescence value of the control group, IF r represents the fluorescence value of the solvent group.

[0050] 3. Statistical analysis

[0051] The data were expressed as mean ± SD, and the t test was used for comparison among the groups. P < 0.05 indicated a significant difference.

[0052] 3. Experimental Results

[0053] Each sample to be tested has Cu 2+ Induction of Aβ 25-35 The inhibition percentage of aggregation is shown in Table 1 (* indicates that compared with comparative sample 1, **P<0.01; # indicates that compared with comparative sample 2, ## P<0.01; & indicates that compared with positive compounds, && P<0.01).

[0054] Table 1 The Cu 2+ Induction of Aβ 25-35 Percent inhibition of aggregation

[0055] Samples to be tested Inhibition percentage (%) Positive compounds 47.55±1.28 Compound (I) <![CDATA[78.12±1.45 **，&& ]]> Compound (II) <![CDATA[80.93±2.56 ##，&& ]]> Comparative Sample 1 56.80±1.17 Comparative Sample 2 52.28±2.49

[0056] The above results show that:

[0057] (1) Compounds (I) and (II) can effectively inhibit Cu 2+ Induction of Aβ 25-35 Aggregation, and the inhibitory effect is significantly stronger than that of the positive compound at the same concentration;

[0058] (2) Comparative sample 1 is a composition of two raw materials for synthesizing compound (I), and its Cu 2+ Induction of Aβ 25-35 The inhibitory effect of the two raw materials on Aβ aggregation was significantly weaker than that of compound (I), indicating that the combination of the two raw materials and compound (I) have the same inhibitory effect on Aβ aggregation. 25-35 There is no comparison in terms of activity of aggregation;

[0059] (3) Comparative sample 2 is a composition of two raw materials for synthesizing compound (II), and its Cu 2+ Induction of Aβ 25-35 The inhibitory effect of the two raw materials on Aβ aggregation was significantly weaker than that of compound (II), indicating that the combination of the two raw materials and compound (II) have the same inhibitory effect on Aβ aggregation. 25-35 There is no comparison in terms of aggregation activity.

[0060] Example 4: Effects of Compounds (I) and (II) on the CL4176 Nematode Paralysis Model

[0061] 1. Experimental Materials

[0062] MgSO₄ was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; agar powder, yeast powder, cholesterol, tryptone, NaCl, and CaCl₂ were all purchased from Beijing Solaibao Technology Co., Ltd.; Na₂HPO₄·12H₂O and KH₂PO₄ were purchased from Sinopharm Chemical Reagent Co., Ltd.; the transgenic AD model Caenorhabditis elegans strain CL4176 [dvIs27[myo-3p::A-Beta(1-42)::let-8513'UTR)+rol-6(su1006)]X] and uracil-deficient Escherichia coli OP50 were purchased from the Caenorhabditis elegans Genetics Center. Compounds (I) and (II) were prepared as in Example 1.

[0063] 2. Experimental Methods

[0064] 1. Solution preparation

[0065] M9 buffer: Weigh 0.6 g of disodium hydrogen phosphate, 0.3 g of potassium dihydrogen phosphate, 0.5 g of sodium chloride, and 0.025 g of magnesium sulfate (dehydrated), dissolve in 100 mL of deionized water, and sterilize at high temperature before use.

[0066] NGM culture plates: Weigh 3.0g sodium chloride, 2.5g tryptone, and 17g agar into a conical flask, add 975mL deionized water, and autoclave. When the sterilization solution temperature drops to approximately 55°C, add 0.5mL 1M calcium chloride solution, 1mL 5mg / mL cholesterol solution, 1mL 1M magnesium sulfate solution, and 25mL 1M potassium phosphate buffer in that order. Use a pipette to add the prepared culture medium to the corresponding dishes or well plates and cool to solidify overnight.

[0067] Sample stock solution to be tested:

[0068] (1) Compound (I) stock solution: Weigh an appropriate amount of Compound (I) and dissolve it in DMSO to prepare a 10 mM solution;

[0069] (2) Compound (II) stock solution: Weigh an appropriate amount of compound (II) and dissolve it in DMSO to prepare a 10 mM solution;

[0070] (3) Comparative sample 1 mother liquor: Weigh appropriate amounts of (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid and 4-(2-benzothiazolyl)aniline and dissolve them in DMSO to prepare a mixed solution of 10 mM (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid + 10 mM 4-(2-benzothiazolyl)aniline;

[0071] (4) Comparative sample 2 mother liquor: Appropriate amounts of (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid and riluzole were weighed and dissolved in DMSO to prepare a mixed solution of 10 mM (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid + 10 mM riluzole.

[0072] 2. Grouping, Modeling, and Drug Administration

[0073] Dilute the stock solution from each group 20-fold with M9 buffer, then evenly spread 500 μL of each 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. Inoculate the drug-containing plates with synchronized Caenorhabditis elegans. A group without drug was also established as a model group.

[0074] The synchronized Caenorhabditis elegans were cultured at 16°C for 48 hours, ie, the Caenorhabditis elegans grew to the L3 stage, and the culture plate was transferred to 25°C to induce transcriptional expression of the Aβ gene.

[0075] The culture plates were transferred to a 25°C incubation temperature, and the number of paralyzed C. elegans was recorded after 36 hours. Paralyzed C. elegans individuals exhibited partial rigidity of the trunk, leading to motor impairment or even loss of motor function, and their heads could twist.

[0076] 4. Detection indicators

[0077] For paralysis rate testing, each group should contain at least 30 nematodes. Paralysis rate within 36 hours (%) = number of paralyzed nematodes / total number of nematodes × 100%. Each experiment was repeated three times.

[0078] 5. Statistical analysis

[0079] The data were expressed as mean ± SD, and the t test was used for comparison among the groups. P < 0.05 indicated a significant difference.

[0080] 3. Experimental Results

[0081] The results of nematode paralysis rate test in each group are shown in Table 2 (* indicates that compared with control sample 1, **P < 0.01; # indicates that compared with control sample 2, ## P<0.01; & indicates that compared with the model group, && P<0.01).

[0082] Table 2 Paralysis rate of nematodes in each group

[0083] Samples to be tested Paralysis rate (%) Model Group 91.7±3.98 Compound (I) <![CDATA[63.97±2.54 **，&& ]]> Compound (II) <![CDATA[58.95±3.09 ##，&& ]]> Comparative Sample 1 86.19±2.07 Comparative Sample 2 85.92±0.73

[0084] The above results show that:

[0085] (1) Compounds (I) and (II) can effectively inhibit paralysis in the AD model of Caenorhabditis elegans, and the paralysis rate is significantly lower than that of the model group;

[0086] (2) Comparative sample 1 is a composition composed of two raw materials for synthesizing compound (I). Its inhibitory effect on paralysis in the AD model Caenorhabditis elegans is significantly weaker than that of compound (I), indicating that the composition composed of the two raw materials is not comparable to compound (I) in terms of anti-paralysis activity in the AD model Caenorhabditis elegans.

[0087] (3) Comparative sample 2 is a composition composed of two raw materials for synthesizing compound (II). Its inhibitory effect on paralysis of AD model Caenorhabditis elegans is significantly weaker than that of compound (II), indicating that the composition composed of two raw materials is not comparable to compound (II) in terms of anti-paralysis activity of AD model Caenorhabditis elegans.

[0088] In summary:

[0089] The present invention provides two compounds with novel structures. The synthesis method of these two new compounds is simple and easy to produce. Anti-Aβ aggregation experiments show that both new compounds have anti-Aβ aggregation activity stronger than that of positive compounds. Those skilled in the art know that Aβ aggregation refers to the process in which β-amyloid protein spontaneously forms aggregates in the brain, and abnormal aggregation of Aβ protein is one of the main pathological characteristics of AD. Aβ protein spontaneously forms aggregates such as oligomers, fibrils and fibers in the brain. These aggregates can cause dysfunction and death of nerve cells, and eventually lead to dementia. The present invention further uses AD model nematodes as a model organism to prove that these two new compounds have a significant effect of inhibiting paralysis in AD model nematodes. Therefore, the two new compounds provided by the present invention have the prospect of being developed into drugs for treating diseases caused by abnormal aggregation of β-amyloid protein, such as AD.

[0090] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A compound having a structural formula as shown in Formula I or II, or a pharmaceutically acceptable salt thereof; 2. A method for preparing the compound of formula I according to claim 1, characterized in that: The raw materials for the preparation include (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid and 4-(2-benzothiazolyl)aniline.

3. The method according to claim 2, wherein: The molar ratio of the two raw materials is 1:

1.

4. A method for preparing the compound of formula II according to claim 1, characterized in that: The preparation raw materials include (2S)-2-(2-oxopyrrolidin-1-yl)butyric acid and riluzole.

5. The method according to claim 4, characterized in that: The molar ratio of the two raw materials is 1:

1.

6. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof for preparing a medicament for treating diseases caused by abnormal aggregation of β-amyloid protein.

7. The use according to claim 6, characterized in that: The disease caused by abnormal aggregation of β-amyloid protein is Alzheimer's disease.

8. A pharmaceutical preparation, characterized in that: The compound according to claim 1 or a pharmaceutically acceptable salt thereof is used as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier or excipient.

9. Use of the pharmaceutical preparation according to claim 8 for preparing a drug for treating diseases caused by abnormal aggregation of β-amyloid protein.

10. The use according to claim 9, characterized in that: The disease caused by abnormal aggregation of β-amyloid protein is Alzheimer's disease.

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