A compound against aβ aggregation and its use in pharmacy

Abstract

This invention discloses a compound that inhibits Aβ aggregation and its application in pharmaceuticals. The novel compound provided by this invention exhibits significantly stronger anti-Aβ aggregation activity than positive control compounds. Aβ protein spontaneously forms aggregates such as oligomers, fibrils, and fibers in the brain. These aggregates lead to neuronal dysfunction and death, ultimately causing neurodegenerative diseases. This invention demonstrates, using a commonly used AD research model in the field, that this novel compound with anti-Aβ aggregation activity possesses significant anti-AD activity.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to the discovery and use of new compounds, specifically to a compound that inhibits Aβ aggregation and its application in pharmaceuticals. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease that severely impacts the quality of life of older adults. Its pathological features primarily include plaques formed by the abnormal deposition of β-amyloid (Aβ), neurofibrillary tangles, and widespread neuronal death. With the increasing global population aging trend, the incidence of AD is rising year by year, becoming one of the major public health challenges facing modern society.

[0003] Currently, treatment for Alzheimer's disease (AD) mainly focuses on symptom relief, and there are no drugs that can cure AD. Therefore, developing new and effective AD treatments is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a compound that inhibits Aβ aggregation and its application in pharmaceuticals.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] A compound of formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof;

[0007]

[0008] The use of one of the above-mentioned compounds or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating diseases caused by abnormal aggregation of β-amyloid protein.

[0009] Preferably, the disease caused by the abnormal aggregation of β-amyloid protein is Alzheimer's disease.

[0010] A pharmaceutical preparation comprising the above-mentioned compound or its pharmaceutically acceptable salt or solvate as the active ingredient, and formulated into a pharmaceutically acceptable dosage form via a pharmaceutically acceptable carrier or excipient.

[0011] The use of one of the above-mentioned pharmaceutical preparations in the preparation of a drug for treating diseases caused by abnormal aggregation of β-amyloid protein.

[0012] Preferably, the disease caused by the abnormal aggregation of β-amyloid protein is Alzheimer's disease.

[0013] Beneficial effects:

[0014] The novel compound provided by this invention exhibits significantly stronger anti-Aβ aggregation activity than positive control compounds. Aβ protein spontaneously forms aggregates such as oligomers, fibrils, and fibers in the brain. These aggregates lead to neuronal dysfunction and death, ultimately causing neurodegenerative diseases. This invention demonstrates, using a commonly used AD research model in the field, that this novel compound with anti-Aβ aggregation activity possesses significant anti-AD activity. Detailed Implementation

[0015] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0016] Example 1: Preparation of compounds (I) and (II)

[0017] The synthesis route is as follows:

[0018]

[0019] (2S)-2-(2-oxopyrrolidone-1-yl)butyric acid (0.5 mmol) and 4-amino-N-(1-isopropylpiperidin-4-yl)benzamide (0.5 mmol) were dissolved in 10 mL of dichloromethane. Then, 1-propyl phosphate cyclic anhydride (T3P, 1.5 mmol) and triethylamine (TEA, 4 mmol) were added. The mixture was stirred at room temperature for 24 h. After the reaction was completed, the dichloromethane was removed by vacuum distillation. Then, 10 mL of ethyl acetate was added, and the mixture was 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 for chromatography. The mixture was eluted isocratically with a 2:1 volume ratio of petroleum ether / ethyl acetate mixture. The mixture was detected by TLC. The eluent corresponding to the target compound was collected, concentrated and dried to obtain the compound with the structure of formula (Ⅰ). 1 HNMR (500MHz, DMSO-d6): 1 H NMR (500MHz, DMSO-d6) δ8.93(s,1H),7.67(m,2H),7.57(m,2H),6.01(d,J=8.1Hz,1H),4.55(t,J=7.8Hz,1H),3.96(s,1H),3.49(m ,2H),2.86(m,2H),2.75(m,1H),2.46(m,2H),2.31(m,3H),2.05(m,5H),1.54(d,2H),1.05(d,J=6.5Hz,6H),0.95(t,J=7.4Hz,3H).

[0020] (2S)-2-(2-oxopyrrolidone-1-yl)butyric acid (0.5 mmol) and 5-methoxytryptamine (0.5 mmol) were dissolved in 10 mL of dichloromethane, and then 1-propyl phosphate cyclic anhydride (T3P, 1.5 mmol) and triethylamine (TEA, 4 mmol) were added. The mixture was stirred at room temperature for 24 h. After the reaction was completed, the dichloromethane was removed by vacuum distillation. Then, 10 mL of ethyl acetate was added, and the mixture was 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 for chromatography. The mixture was eluted isocratically with a 2:1 volume ratio of petroleum ether / ethyl acetate mixture. The mixture was detected by TLC, and the eluent corresponding to the target compound was collected, concentrated and dried to obtain the compound with the structure of formula (II). 1 H NMR(500MHz,DMSO-d6)δ8.11(s,1H),7.24(m,1H),7.01(m,2H),6.85(m,1H),6.19(s,1H),4.32(m,1H),3.86(s,3H),3.62–3.51(m,2H),3.32 –3.20(m,2H),2.95(m,1H),2.91(m,2H),2.88(m,1H),2.36–2.29(m,1H ), 2.15(m,1H),1.97–1.88(m,1H),1.64(m,1H),0.84(t,J=7.4Hz,3H).

[0021] Example 2: Compounds (I) and (II) on Cu 2+ Inducing Aβ 25-35 The effects of aggregation

[0022] I. Experimental Materials

[0023] Aβ 25-35 Compounds (I) and (II) were purchased from Beijing Bio-Sens Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) and thioflavin T (ThT) were purchased from Shanghai E. En Chemical Technology Co., Ltd. Compounds (I) and (II) were prepared according to Example 1. The experimental water was self-prepared ultrapure water.

[0024] II. Experimental Methods

[0025] 1. Solution preparation

[0026] HEPES buffer: Weigh an appropriate amount of HEPES and dissolve it in ultrapure water to prepare a 10mM buffer (pH 7.2).

[0027] Thioflavin T (ThT) solution: Weigh an appropriate amount of thioflavin T, dissolve it in HEPES buffer, and prepare an 80 μM solution.

[0028] Cu 2+Solution: Weigh an appropriate amount of CuCl2, dissolve it in HEPES buffer, and prepare a 200 μM solution.

[0029] Aβ 25-35 Solution: Weigh an appropriate amount of Aβ 25-35 Dissolve in HEPES buffer (containing 37.5% DMSO) to prepare a 500 μM solution.

[0030] The mother liquor of the sample to be tested:

[0031] (1) Positive compound stock solution: Weigh an appropriate amount of curcumin and dissolve it in DMSO to prepare a 200 μM solution;

[0032] (2) Mother liquor of compound (I): Weigh an appropriate amount of compound (I) and dissolve it in DMSO to prepare a 200 μM solution;

[0033] (3) Mother liquor of compound (II): Weigh an appropriate amount of compound (II) and dissolve it in DMSO to prepare a 200 μM solution;

[0034] (4) Comparison of sample 1 stock solution: Weigh an appropriate amount of 4-amino-N-(1-isopropylpiperidin-4-yl)benzamide and (2S)-2-(2-oxopyrrolidone-1-yl)butyric acid and dissolve them in DMSO to prepare a 200 μM mixed solution of 4-amino-N-(1-isopropylpiperidin-4-yl)benzamide + 200 μM (2S)-2-(2-oxopyrrolidone-1-yl)butyric acid;

[0035] (5) Comparison of sample 2 mother liquor: Weigh an appropriate amount of 5-methoxytryptamine and (2S)-2-(2-oxopyrrolidone-1-yl)butyric acid and dissolve them in DMSO to prepare a mixed solution of 200 μM 5-methoxytryptamine + 200 μM (2S)-2-(2-oxopyrrolidone-1-yl)butyric acid.

[0036] 2. Grouping, modeling, and drug administration

[0037] Experimental group: 166 μL HEPES buffer, 10 μL Cu 2+ Solution, 4 μL Aβ 25-35 Solution, 10 μL of the stock solution of the sample to be tested.

[0038] Control group: 170 μL HEPES buffer, 10 μL Cu 2+ Solution, 10 μL of the stock solution of the sample to be tested.

[0039] Control group: 166 μL HEPES buffer, 10 μL Cu 2+ Solution, 4 μL Aβ 25-35 Solution, 10 μL LDMSO solution.

[0040] Solvent group: 170 μL HEPES buffer, 10 μL Cu 2+ Solution, 10 μL LDMSO solution.

[0041] Add the solution to black 96-well plates, with three 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 values ​​using a multi-functional microplate reader at excitation wavelength of 440 nm and emission wavelength of 480 nm. Calculate the effect of the sample on Cu using the following formula. 2+ Inducing Aβ 25-35 Percentage of aggregation inhibition.

[0042] Inhibition percentage (%) = [1-(IF)] i -IF0) / (IF c -IF r )]×100%.

[0043] IF in the formula i IF represents the fluorescence value of the experimental group, IF0 represents the fluorescence value of the blank group, and IF c The fluorescence value representing the control group, IF r The fluorescence value represents the solvent group.

[0044] 3. Statistical Analysis

[0045] Data are expressed as mean ± SD. The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0046] III. Experimental Results

[0047] Each sample to be tested for Cu 2+ Inducing Aβ 25-35 The percentage of aggregation inhibition is shown in Table 1 (* indicates that compared with control sample 1, **P<0.01; # This indicates that compared to control sample 2, ## P < 0.01; & This indicates that compared to positive compounds, && As shown in the figure (P < 0.01).

[0048] Table 1. Effects of each sample on Cu 2+ Inducing Aβ 25-35 Percentage of aggregation inhibition

[0049] Sample to be tested Inhibition percentage (%) Positive compounds 46.02±2.05 Compound (I) <![CDATA[68.43±3.26 **，&& ]]> Compound (II) <![CDATA[73.09±3.25 ##，&& ]]> Comparison Sample 1 39.60±3.41 Comparison Sample 2 43.38±2.59

[0050] The above results indicate that:

[0051] (1) Both compounds (I) and (II) can effectively inhibit Cu 2+ Inducing Aβ 25-35Aggregation, and the inhibitory effect is significantly stronger than that of positive compounds at the same concentration;

[0052] (2) Comparison sample 1 is a composition of two raw materials for synthesizing compound (I), and its Cu 2+ Inducing Aβ 25-35 The inhibitory effect on aggregation was significantly weaker than that of compound (I), indicating that the composition of the two raw materials and compound (I) have similar inhibitory effects on Aβ. 25-35 There is no comparability in terms of aggregation activity;

[0053] (3) Comparison sample 2 is a composition of two raw materials for synthesizing compound (II), and its Cu 2+ Inducing Aβ 25-35 The inhibitory effect on aggregation was significantly weaker than that of compound (II), indicating that the composition of the two raw materials and compound (II) are similar in their anti-Aβ activity. 25-35 There is no comparability in terms of aggregation activity.

[0054] Example 3: Effects of compounds (I) and (II) on the paralysis model of CL4176 nematode

[0055] I. Experimental Materials

[0056] MgSO4 was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; agar powder, yeast powder, cholesterol, tryptone, NaCl, and CaCl2 were all purchased from Beijing Solarbio Science & Technology Co., Ltd.; Na2HPO4·12H2O and KH2PO4 were both purchased from Sinopharm Chemical Reagent Co., Ltd.; the transgenic AD model *C. elegans* strain CL4176[dvIs27[myo-3p::A-Beta(1-42)::let-8513'UTR)+rol-6(su1006)]X] and the uracil-deficient *Escherichia coli* OP50 were both purchased from the *C. elegans* Genetic Center in the United States. Compounds (I) and (II) were prepared according to Example 1.

[0057] II. Solution Preparation

[0058] 1. Solution preparation

[0059] M9 buffer: Weigh 0.6g disodium hydrogen phosphate, 0.3g potassium dihydrogen phosphate, 0.5g sodium chloride, and 0.025g magnesium sulfate (dehydrated), dissolve in 100mL deionized water, sterilize at high temperature, and set aside for use.

[0060] NGM culture plate: Weigh 3.0g sodium chloride, 2.5g tryptone, and 17g agar into an Erlenmeyer flask, add 975mL deionized water, and autoclave. When the temperature of the sterilized solution drops to approximately 55℃, 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 solution in sequence. Use a pipette to add the prepared culture medium liquid to the corresponding petri dishes or well plates, and allow to cool and solidify overnight.

[0061] The mother liquor of the sample to be tested:

[0062] (1) Mother liquor of compound (I): Weigh an appropriate amount of compound (I) and dissolve it in DMSO to prepare a 10 mM solution;

[0063] (2) Mother liquor of compound (II): Weigh an appropriate amount of compound (II) and dissolve it in DMSO to prepare a 10 mM solution;

[0064] (4) Comparison of sample 1 mother liquor: Weigh an appropriate amount of 4-amino-N-(1-isopropylpiperidin-4-yl)benzamide and (2S)-2-(2-oxopyrrolidone-1-yl)butyric acid and dissolve them in DMSO to prepare a mixed solution of 10mM 4-amino-N-(1-isopropylpiperidin-4-yl)benzamide + 10mM (2S)-2-(2-oxopyrrolidone-1-yl)butyric acid;

[0065] (5) Comparison of sample 2 mother liquor: Weigh an appropriate amount of 5-methoxytryptamine and (2S)-2-(2-oxopyrrolidone-1-yl)butyric acid and dissolve them in DMSO to prepare a mixed solution of 10mM 5-methoxytryptamine + 10mM (2S)-2-(2-oxopyrrolidone-1-yl)butyric acid.

[0066] 2. Grouping, modeling, and drug administration

[0067] Each group's stock solution was diluted 20-fold with M9 buffer, and 500 μL of each solution was evenly spread onto freshly prepared NGM plates and allowed to air dry at room temperature. 50 μL of OP50 bacterial suspension was added to the center of each plate, and the plates were dried overnight. Synchronized *C. elegans* were then inoculated onto the drug-containing plates. A group without the drug was also set up as a model group.

[0068] The synchronized Caenorhabditis elegans were cultured at 16°C for 48 hours, which is when the Caenorhabditis elegans grew to the L3 stage. The culture plate was then transferred to 25°C to induce the transcriptional expression of the Aβ gene.

[0069] The culture plates were transferred to 25°C for incubation, and the number of paralyzed *C. elegans* was recorded after 36 hours. The paralyzed *C. elegans* individuals exhibited stiffness in the trunk, leading to motor impairment or even loss of motor function; their heads could be twisted.

[0070] 3. Detection indicators

[0071] In the paralysis rate detection experiment, the number of nematodes in each group was no less than 30. The nematode paralysis rate % at 36 hours was calculated as: (Number of paralyzed nematodes / Total number of nematodes) × 100%. Each experiment was repeated 3 times.

[0072] 4. Statistical Analysis

[0073] Data are expressed as mean ± SD. The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0074] III. Experimental Results

[0075] The results of the nematode paralysis rate detection in each group are shown in Table 2 (* indicates that compared with the control sample 1, *P<0.05; # This indicates that compared to control sample 2, # P < 0.05; & indicates that compared to the model group, && As shown in the figure (P < 0.01).

[0076] Table 2. Nematode paralysis rate in each group

[0077] Sample to be tested Paralysis rate (%) Model group 95.54±1.45 Compound (I) <![CDATA[75.41±3.34 *，&& ]]> Compound (II) <![CDATA[71.36±3.55 #，&& ]]> Comparison Sample 1 90.69±1.18 Comparison Sample 2 87.84±2.49

[0078] The above results indicate that:

[0079] (1) Both compounds (I) and (II) can effectively inhibit paralysis in the AD model of Caenorhabditis elegans, and the paralysis rate is significantly lower than that in the model group.

[0080] (2) The composition of the two raw materials of the synthetic compound (I) in comparison sample 1 showed that its inhibitory effect on the paralysis of the AD model Caenorhabditis elegans was significantly weaker than that of compound (I), indicating that the composition of the two raw materials and compound (I) were not comparable in terms of their activity against the paralysis of the AD model Caenorhabditis elegans.

[0081] (3) Comparative sample 2 is a combination of two raw materials of synthetic compound (II). Its inhibitory effect on paralysis of AD model Caenorhabditis elegans is significantly weaker than that of compound (II), indicating that the combination of the two raw materials is not comparable to compound (II) in terms of activity against paralysis of AD model Caenorhabditis elegans.

[0082] In summary, the novel compound provided by this invention exhibits significantly stronger anti-Aβ aggregation activity than positive control compounds. Aβ protein spontaneously forms aggregates such as oligomers, fibrils, and fibers in the brain. These aggregates lead to neuronal dysfunction and death, ultimately causing neurodegenerative diseases. This invention demonstrates, using a commonly used AD research model in the field, that this novel compound with anti-Aβ aggregation activity possesses significant anti-AD activity.

[0083] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. A compound of formula (I) or formula (II), or a pharmaceutically acceptable salt or solvate thereof; 2. The use of the compound of claim 1 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating diseases caused by abnormal aggregation of β-amyloid protein.

3. The application according to claim 2, characterized in that: The disease caused by the abnormal aggregation of β-amyloid protein is Alzheimer's disease.

4. A pharmaceutical preparation, characterized in that: Using the compound of claim 1 or its pharmaceutically acceptable salt or solvate as the active ingredient, a pharmaceutically acceptable dosage form is prepared using a pharmaceutically acceptable carrier or excipient.

5. The use of the pharmaceutical preparation of claim 4 in the preparation of a medicament for treating diseases caused by abnormal aggregation of β-amyloid protein.

6. The application according to claim 5, characterized in that: The disease caused by the abnormal aggregation of β-amyloid protein is Alzheimer's disease.

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