阅读说明：本技术 抑制淀粉样β蛋白聚集的小分子化合物和制备方法及其应用 (Small molecular compound for inhibiting amyloid beta protein aggregation and preparation method and application thereof ) 是由 董晓燕 许少莹 余林玲 孙彦 于 2021-07-21 设计创作，主要内容包括：本发明涉及抑制淀粉样β蛋白聚集的小分子化合物和制备方法及其应用。利用3,4,5-三羟基苯甲酸表面羧基偶联谷氨酰胺,获得4-氨基甲酰基-2-[(3,4,5-三羟基苯基)甲酰氨基]丁酸化合物。该小分子化合物在5-25μM的浓度范围内能够有效抑制淀粉样β蛋白的聚集,降低β折叠结构的含量,清除阿尔兹海默症模型秀丽隐杆线虫CL2006体内的淀粉样斑块,并CL2006的寿命延长了三分之一。与同类的小分子类和异类的多肽或蛋白类阿尔兹海默症抑制剂相比具有显著的淀粉样蛋白抑制效果和良好的血脑屏障穿透性；能够改变Aβ-(42)的聚集形态,抑制淀粉样蛋白纤维的生成。作为制备治疗阿尔茨海默氏症的药物应用具有广阔前景。(The invention relates to a small molecular compound for inhibiting amyloid beta protein aggregation, a preparation method and application thereof. Coupling glutamine by utilizing carboxyl on the surface of 3,4, 5-trihydroxybenzoic acid to obtain 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido]And a butyric acid compound. The small molecular compound can effectively inhibit the aggregation of amyloid beta protein within the concentration range of 5-25 mu M, reduce the content of beta folding structure, eliminate amyloid plaques in the body of the Alzheimer's disease model C.elegans CL2006, and prolong the life of the CL2006 by one third. Compared with similar micromolecule and heterogeneous polypeptide or protein Alzheimer disease inhibitor, the inhibitor has obvious amyloid protein inhibition effect and good blood brain barrier penetrability; can change A beta 42 Inhibiting amyloid proteinAnd (5) generation of white fibers. Has wide application prospect when being used for preparing the medicine for treating the Alzheimer disease.)

1. A small molecule compound for inhibiting amyloid beta protein aggregation is characterized in that carboxyl of 3,4, 5-trihydroxybenzoic acid reacts with main chain amino of glutamine to obtain 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido ] butyric acid, and the structural formula is as follows:

2. a method for preparing a small molecule compound that inhibits amyloid beta protein aggregation according to claim 1, characterized in that: modifying glutamine on carboxyl of 3,4, 5-trihydroxybenzoic acid, connecting carboxyl of 3,4, 5-trihydroxybenzoic acid with amino of glutamine main chain, and keeping carboxyl of glutamine main chain in free state to obtain small molecule compound 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido ] butyric acid compound with stable structure.

3. The small molecule compound for inhibiting amyloid beta protein aggregation according to claim 1, wherein the 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido ] butanoic acid compound has an effect of inhibiting amyloid beta protein aggregation.

4. The small molecule compound for inhibiting amyloid β protein aggregation according to claim 1, characterized in that the 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido ] butanoic acid compound has an effect of clearing amyloid β protein aggregation in c.

5. The small molecule compound for inhibiting amyloid beta protein aggregation according to claim 1, wherein the 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido ] butanoic acid compound has the ability to cross the blood brain barrier.

6. The small molecule compound for inhibiting amyloid beta protein aggregation according to claim 1, wherein the inhibitory effect of the 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido ] butanoic acid compound is superior to that of the related small molecule drugs and the related polypeptide or protein drugs.

7. The small molecule compound for inhibiting amyloid beta protein aggregation according to claim 1, wherein the blood brain barrier penetration of the 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido ] butanoic acid compound is superior to that of the related small molecule drugs and the related polypeptide or protein drugs.

8. The small molecule compound for inhibiting amyloid beta protein aggregation according to claim 1, characterized by the use of a 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido ] butanoic acid compound as a medicament for the treatment of alzheimer's disease.

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a small molecular compound for inhibiting amyloid beta protein aggregation, a preparation method and application thereof.

Background

Alzheimer's Disease (AD) is a neurodegenerative disease. In recent years, AD has become a global health problem, and the incidence rate of AD is increased with the aging of the world population, about 5000 million people are affected globally, and the number of the people is predicted to increase to three times of the existing disease by 2050 (Lancet,2021,397: 1577-1590). The clinical manifestations of AD are loss of memory and cognitive function, with the main pathological features being extracellular senile plaque deposits and intracellular neurofibrillary tangles (Nature,2014,515: 274-278). Wherein the senile plaque is formed by the aggregation of Amyloid beta protein (A beta) in brain tissue. A beta is produced by hydrolysis of Amyloid Precursor Protein (APP) by beta-and gamma-secretases, and A beta is mainly present in vivo₄₀And Abeta₄₂Two forms, wherein A beta₄₀It is abundant in cerebrospinal fluid, about 90%, and Abeta₄₂More easily aggregated and more toxic (Journal of biological chemistry,2012,287: 5650-. The amyloid cascade hypothesis states that the A β monomers fold continuously from an irregular coil structure into an ordered β -sheet structure, which subsequently aggregate to form oligomers, fibrils and mature fibers, leading to amyloid deposition and neurotoxicity (Science,2002,297: 353-. Most of the currently developed AD therapeutic drugs have adverse reactions in clinical trials, such as hepatotoxicity or skin cancer, and some drugs can only delay the symptoms of AD patients and cannot prevent and completely cure AD (ACS chemical neuroscience,2018,9: 198-. Therefore, it is important to develop an inhibitor having good biocompatibility and capable of effectively inhibiting a β aggregation.

Existing inhibitors can be divided into four major classes, including polypeptide inhibitors, protein inhibitors, nano-inhibitors, and small molecule inhibitors. The polypeptide inhibitor has high specificity and is easy to modify, but the polypeptide is easy to self-aggregate, and the effective concentration is higher (ACS chemical neuroscience,2019,10: 1390-. Protein inhibitors have good biocompatibility and are convenient for chemical modification, but have a large molecular weight and poor blood brain barrier penetration (Journal of organic biochemical, 2017,171: 67-75). The nano inhibitor has large specific surface area, is easy for surface modification, but has poor biocompatibility and low specificity (ACS applied materials & interfaces,2015,7: 5650-. In contrast, small molecule inhibitors have simple structure, strong antioxidant capacity, low effective concentration, and good blood brain barrier penetration, and are widely noticed by researchers (Science of the total environment,2020,725: 138313).

Polyphenols are widely found in food, and various studies have shown that natural polyphenols inhibit the formation of amyloid fibrils and exhibit good resistance to amyloidosis (Molecular differentiation & food research,2015,59: 8-20). Epigallocatechin gallate (EGCG) is the main component of green tea, and can significantly inhibit the formation of Abeta aggregates. EGCG can bind directly to A beta, interfering with the formation of an ordered beta-sheet structure during A beta aggregation, and forming non-toxic spherical aggregates (Nature structural & molecular biology,2008,15: 558-566). 3,4, 5-trihydroxybenzoic acid (GA) as a key component of EGCG was also able to inhibit the aggregate deposition of A β and its induced neurotoxicity in the brains of AD transgenic mice (Molecular differentiation & food research,2011,55: 1798) -1808). In addition, GA has antioxidant, anti-inflammatory, antiviral and anticancer activities (Biological & pharmacological bulletin,2007,30: 1052-. As can be seen from comparison in kinetic experiments, the GA inhibitory effect is higher than that of EGCG at the same mass concentration. Therefore, it is presumed that a carboxyl group specific to GA is crucial for polyphenol having high structure dependence.

The invention is designed based on GA, carboxyl of GA is combined with main chain amino of glutamine to obtain 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido ] butyric acid compound (4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido ] butanoic acid, CTFBA), CTFBA and A beta have higher affinity, and the aggregation of A beta can be effectively inhibited under low concentration.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, synthesize a small molecular compound with the function of inhibiting amyloid beta protein aggregation, and provide a preparation method of the small molecular compound and application of the small molecular compound in inhibiting beta-amyloid protein aggregation.

The 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido ] butyric acid compound not only has obvious inhibition effect on A beta aggregation, but also has better inhibition effect on amyloid beta protein aggregation than similar small molecules. The 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido ] butyric acid compound can also eliminate amyloid plaques in bodies of AD model caenorhabditis elegans CL2006 and prolong the service life of the nematodes, and meanwhile, the 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido ] butyric acid compound has the capability of penetrating blood brain barriers and has better effect than polypeptide and protein inhibitors.

The invention is realized by the following technical means:

a small molecule compound for inhibiting amyloid beta protein aggregation is prepared by reacting carboxyl of 3,4, 5-trihydroxybenzoic acid with main chain amino of glutamine to obtain 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido ] butyric acid compound (4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido ] butanoic acid, CTFBA). The reaction formula is as follows:

the small molecular compound provided by the invention has the function of inhibiting amyloid beta protein aggregation, the preparation method and the application thereof in inhibiting amyloid beta protein aggregation, and has the following advantages:

first, the small molecule compound 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido]The butyric acid compound can effectively inhibit A beta in the concentration range of 5-25 mu M₄₂Significantly reduce A beta₄₂And (3) generation of a beta-sheet structure in the aggregation process.

Second, the small molecule compound 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido]Butyric acid formationThe compound can change A beta₄₂Inhibiting the formation of amyloid fibrils.

Thirdly, the small molecular compound 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido ] butyric acid compound can inhibit the formation of amyloid plaques in the body of the AD model C.elegans CL2006 and eliminate the dyskinesia of the CL 2006.

Fourthly, the small molecular compound 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido]Butyric acid compounds capable of inhibiting A beta expressed in CL2006 in vivo₄₂Aggregation-induced toxicity and one-third prolongation of CL2006 Life is A β₄₂An ideal inhibitor of aggregation.

Fifthly, the small molecular compound 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido]Butyric acid compounds capable of converting Abeta₄₂Oligomeric antibodies A11 and A β₄₂The immunoreaction activity of the fiber antibody OC is rapidly reduced, and A beta can be obviously inhibited₄₂Formation of oligomers and fibers during aggregation.

Sixthly, the small molecule compound 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido]Butyric acid compound p-Abeta₄₂The aggregation inhibition effect is stronger than that of the similar small molecular inhibitor EGCG and better than that of the heterogeneous polypeptide inhibitor LK7 and the protein inhibitor BSA-B.

Seventh, the small molecular compound 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido ] butyric acid compound is not only stronger than the polypeptide inhibitor LK7 and the protein inhibitor BSA-B which do not have blood brain barrier penetrating ability, but also better than the small molecular inhibitor EGCG which also has blood brain barrier penetrating ability, thereby showing that CTFBA has good blood brain barrier penetrating ability.

Drawings

FIG. 1: different concentrations of CTFBA and A β in example 1₄₂ThT fluorescence profiles of the cultures after 48h of co-cultivation.

FIG. 2: EXAMPLE 2 moderate molar ratios of CTFBA to A β₄₂Topography of the culture after 48h of co-cultivation.

FIG. 3: fluorescence microscopy of CTFBA in example 3 for clearing amyloid plaques in C.elegans CL 2006.

FIG. 4: survival of CL2006 in example 4 after co-culture of CTFBA with C.elegans CL 2006.

FIG. 5: CTFBA vs. Abeta in example 5₄₂Inhibition of the immunological activity of oligomer antibody a11 and fiber antibody OC.

FIG. 6: graph comparing CTFBA with ThT fluorescence intensity of congenic and heterogeneous a β aggregation inhibitors.

FIG. 7: comparative figures for the permeability of CTFBA to the blood brain barrier of homogeneous and heterogeneous a β aggregation inhibitors.

Detailed Description

The present invention will be further described with reference to the following specific examples.

The invention couples glutamine on carboxyl of 3,4, 5-trihydroxybenzoic acid to prepare 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido]Butyric acid Compound (CTFBA). A plurality of experimental means prove that 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido]The butyric acid compound can obviously inhibit A beta at the concentration higher than 5 mu M₄₂Eliminates amyloid deposition in the AD model caenorhabditis elegans CL2006, and can prolong the survival of CL 2006. Furthermore, 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido]The butyric acid compound has good blood brain barrier permeability. 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido]Butyric acid compounds inhibit a β₄₂The ability to aggregate is determined by methods such as ThT fluorescence and atomic force microscopy, and its ability to clear amyloid deposits in CL2006 and extend CL2006 survival is determined by fluorescence microscopy experiments and life cycle experiments, 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) carboxamido]The blood brain barrier permeability of the butyric compounds was determined by a parallel artificial membrane permeation method.

Example 1: different concentrations of CTFBA and Abeta₄₂ThT fluorescence intensity of the culture after 48h of co-cultivation.

Mixing A beta with the purity of 95 percent₄₂Dissolving in Hexafluoroisopropanol (HFIP) at a concentration of 1.0mg/mL, ultrasonic treating in ice bath for 30min to break the formed aggregates, and heating at 4 deg.CStanding for 2 hr for dissolving, centrifuging at rotation speed of 16000g at 4 deg.C for 20min, and freezing 75% of the supernatant in-70 deg.C refrigerator overnight. Finally, the A beta is put into a freeze dryer to be dried₄₂Freeze-drying into cotton-like powder, and storing in a refrigerator at-20 deg.C.

Freeze-drying Abeta₄₂Dissolving in 20mM NaOH solution, and ultrasonic treating in ice bath for 10min to obtain 275 μ M Abeta₄₂And (4) mother liquor. Diluted with HEPES buffer (20mM, pH 7.4, containing 100mM NaCl) containing 27.5. mu.M ThT to give A.beta.at a final concentration of 25. mu.M₄₂Solution, as a control experiment.

CTFBA was weighed and dissolved in 20mM HEPES buffer (pH 7.4, containing 100mM NaCl) to obtain inhibitor solutions at concentrations of 2.75,2.75,5.5,13.75 and 27.5. mu.M, respectively. Taking Abeta with the concentration of 275 mu M₄₂The mother liquor was diluted with inhibitor solutions of different concentrations to give final concentrations of 1.25,2.5,5,12.5 and 25. mu.M inhibitor and 25. mu.M A.beta.₄₂And (3) solution. These solutions of different concentrations were incubated for 48h at 37 ℃ and 150rpm together with the control solution.

ThT solution was prepared by weighing 0.80mg of ThT in 100mL of HEPES buffer (20mM, pH 7.4, containing 100mM NaCl) to a final concentration of 25. mu.M. 200 mu L of a sample cultured for 48h is mixed with 2mL of 25 mu M ThT solution, the fluorescence intensity is detected under the excitation wavelength of 440nm and the emission wavelength of 480nm, each group is performed for three times of paralleling, the excitation and emission gap width is 5nm, and the scanning speed is 100 nm/min. The fluorescence intensity at 480nm for different concentrations of inhibitor was plotted as shown in FIG. 1.

As can be seen from FIG. 1, CTFBA is effective in inhibiting A.beta.₄₂And is concentration-dependent, with an optimal concentration range of 5-25 μ M.

Example 2: CTFBA to Abeta₄₂Influence of aggregation morphology.

Ass preparation by the method of example 1₄₂The mother liquor was diluted with HEPES buffer (20mM, pH 7.4, containing 100mM NaCl) to give A.beta.at a final concentration of 25. mu.M₄₂And (3) solution. Reconstitution of A β with 25 μ M CTFBA₄₂Solution of A beta₄₂The final concentration of (3) was 25. mu.M. The solution was heated at 37 ℃ and 1The culture was carried out at 50 rpm. After 48h of incubation, 20. mu.L of the solution was dropped onto a mica plate and allowed to stand for 10min to allow the sample to be sufficiently bound to the mica surface. Then slowly washing with deionized water for 7 times, removing salt ions in the buffer solution, standing and airing. The observation was carried out in the tapping mode of an atomic force microscope (CSPM5500, original). As shown in fig. 2.

As can be seen in FIG. 2A, A β₄₂After 48h of single culture, dense fibrous aggregates were formed, CTFBA and Abeta in FIG. 2B₄₂After co-culture, the fibers almost completely disappeared and a small amount of amorphous aggregates was formed, indicating that CTFBA was able to significantly reduce a β₄₂Production of fiber and modification of Abeta₄₂The aggregate form of (1).

Example 3: CTFBA has clearing effect on amyloid plaques in C.elegans CL 2006.

Firstly, preparing an NMG culture medium, weighing 17g of agar powder, 2.5g of peptone and 3g of sodium chloride, adding the agar powder, the peptone and the sodium chloride into a conical flask, adding deionized water to 1L, sterilizing at 120 ℃ for 20 minutes at high temperature, cooling to 70 ℃, adding 25mL of 1M potassium dihydrogen phosphate, 1mL of 1M magnesium sulfate, 1mL of 5mg/mL cholesterol and 1mL of 1M calcium chloride into the conical flask in sequence, shaking uniformly, introducing into a flat plate, and airing for later use.

Preparing an LB liquid culture medium, weighing 2g of peptone, 1g of yeast powder and 2g of sodium chloride, adding into a conical flask, adding deionized water to 200mL, selecting escherichia coli OP50 to the LB liquid culture medium, and culturing in a shaking table for 12h at 37 ℃ and 220 rpm. And (3) coating the cultured OP50 bacterial liquid into an NGM culture medium, dripping 200 mu L of bacterial liquid into each plate, and inversely placing the plates into a refrigerator at 4 ℃ for later use after the bacterial liquid is dried.

200 μ L of 25 μ M CTFBA was added to NGM medium with OP50, and after the liquid was dried, 10L 4 stage AD model c.elegans mutant CL2006 were picked up in petri dishes and a corresponding blank control without CTFBA was set. After 3 days of culture, the nematodes were washed out of the dishes using 4% tissue cell fixative, fixed at 4 ℃ for 24h, and stained with 10 μ M ThT solution for 4 h. The stained nematodes were placed on a glass slide and observed using an inverted fluorescence microscope (TE2000-U, Nikon, Japan). As shown in fig. 3.

As can be seen in fig. 3A, distinct fluorescent spots appear in mutant CL2006, indicating the generation of a large number of amyloid plaques. Fig. 3B shows that a large number of amyloid plaques in CL2006 were eliminated when 25 μ M CTFBA was added, indicating that CTFBA was able to inhibit amyloid plaque formation in CL 2006.

Example 4: effect of CTFBA on survival of caenorhabditis elegans CL 2006.

NGM medium with OP50 was prepared as in example 3, and 200. mu.L of 25. mu.M CTFBA was inoculated into the medium and a corresponding blank control without CTFBA was set. Addition of 300. mu.L of 150. mu.M each of 5-fluoro-2' -deoxyuridine to the medium inhibited nematode oviposition. After the liquid was dried, 60L 4 stage CL2006 nematodes were picked up into the dishes, and the number of nematodes surviving in the dishes was recorded daily until all nematodes died. To ensure adequate food supply, the plate was rotated every 3 days. As shown in fig. 4.

As can be seen from FIG. 4, the survival cycle of the mutant CL2006 cultured alone is about 12 days, when 25 μ M CTFBA is added, the mortality rate of CL2006 is obviously reduced from the initial stage of culture, and nematodes die completely until day 16, which indicates that CTFBA can reduce the paralysis rate of CL2006, eliminate the dyskinesia of CL2006, and inhibit A β expressed in CL2006 body₄₂Aggregation-induced cytotoxicity, thereby extending the life of CL2006 by one-third.

Example 5: CTFBA to Abeta₄₂Inhibition of the immunological Activity of oligomer antibody A11 and fiber antibody OC

Ass preparation by the method of example 1₄₂Solution of A beta₄₂Separately (25. mu.M) or co-cultured with CTFBA (1.25. mu.M, 5. mu.M, 25. mu.M) at different concentrations for 0h and 48h, 10. mu.L of each was dropped on a nitrocellulose membrane (0.2. mu.m), and left to dry at room temperature. Shake with 10% skim milk for 1h to block nonspecific protein binding on nitrocellulose membrane. The membranes were then washed (5min × 3) with TBST buffer (20mM Tris-HCl,150mM NaCl, 0.05% Tween 20, pH 7.4) and primary antibody 6E10 (1:5000 for all species of a β), a11 (1: 2000 for a β oligomers) and O were added separatelyC (for A β fibers, 1:3000) was shaken at room temperature for 1h and the membrane was washed with TBST (5 min. times.3). Wherein, because the sensitivity of the commercialized primary antibody A11 is low, the sample amount on the nitrocellulose membrane is increased, 100 mu L of sample is taken and dropped on the membrane by 10 times, and 10 mu L of sample is dropped on each time. Subsequently, the membrane was washed with horseradish peroxidase-labeled secondary antibodies, goat anti-rabbit IgG (1:5000) and goat anti-mouse IgG (1:1000) for 1h at room temperature (5 min. times.3) with TBST to remove unbound secondary antibodies. And finally, developing by using an ECL chemiluminescence kit.

As can be seen from FIG. 5A, at 0h, A β₄₂The immunoreactivity of the oligomer antibody A11 is slightly positive, and after 48 hours of culture, the A beta alone₄₂The immune response activity of the compound is rapidly enhanced, which indicates that the Abeta is₄₂More oligomers are produced during aggregation. When CTFBA with different concentrations is added for co-culture, the CTFBA can rapidly reduce the immunoreaction activity of A11, and the CTFBA can remarkably inhibit A beta₄₂Formation of oligomers during aggregation. As can be seen from FIG. 5B, CTFBA enables A β₄₂The immunoreaction activity of the fiber antibody OC is rapidly weakened, and as the concentration of CTFBA is increased, the blotch is gradually lightened to almost disappear, which shows that CTFBA can obviously inhibit A beta₄₂And (5) generating fibers.

Example 6: comparison of CTFBA with ThT fluorescence intensity of similar and heterogeneous A beta aggregation inhibitors

Ass preparation by the method of example 1₄₂The mother liquor was diluted with HEPES buffer (20mM, pH 7.4, containing 100mM NaCl) to give A.beta.at a final concentration of 25. mu.M₄₂And (3) solution. Weighing small molecule CTFBA, similar small molecule inhibitor EGCG, polypeptide inhibitor LVFFARK (LK7) and protein inhibitor alkalized bovine serum albumin (BSA-B), respectively dissolving in 20mM HEPES buffer solution (pH 7.4, containing 100mM NaCl) to obtain an inhibitor solution with the concentration of 27.5. mu.M. Taking Abeta with the concentration of 275 mu M₄₂The mother liquor was diluted with 27.5. mu.M inhibitor solution to obtain a final concentration of 25. mu.M inhibitor and 25. mu.M A.beta.₄₂And (3) solution. These solutions of different concentrations were incubated for 48h at 37 ℃ and 150rpm together with the control solution.

ThT solution was prepared by weighing 0.80mg of ThT in 100mL of HEPES buffer (20mM, pH 7.4, containing 100mM NaCl) to a final concentration of 25. mu.M. 200 mu L of a sample cultured for 48h is mixed with 2mL of 25 mu M ThT solution, the fluorescence intensity is detected under the excitation wavelength of 440nm and the emission wavelength of 480nm, each group is performed for three times of paralleling, the excitation and emission gap width is 5nm, and the scanning speed is 100 nm/min. The fluorescence intensity at 480nm for different concentrations of inhibitor was plotted as shown in FIG. 6.

As can be seen from FIG. 6, CTFBA at 25. mu.M was able to effectively inhibit A.beta.₄₂The CTFBA has the inhibiting effect which is stronger than that of the similar small-molecule inhibitor EGCG and better than that of the heterogeneous polypeptide inhibitor LK7 and the protein inhibitor BSA-B, so that the CTFBA is a very potential small-molecule AD medicament.

Example 7: comparison of CTFBA with similar and heterogeneous a β aggregation inhibitors for permeability of the blood brain barrier.

The blood brain barrier permeability of the inhibitor CTFBA with similar and heterogeneous a β aggregation inhibitors was determined by parallel artificial membrane penetration (PAMPA-BBB). Small molecule CTFBA, similar small molecule inhibitor EGCG, polypeptide inhibitor LVFFARK (LK7) and protein inhibitor alkalized bovine serum albumin (BSA-B) are respectively dissolved in PBS/EtOH (v/v,7/3) buffer solution to obtain a final concentration of 25 μ M. To the lower 96 well receiver plate was added 300. mu.L PBS/EtOH buffer, to the middle filter was added 4. mu.L of porcine brain lipids dissolved at 20mg/mL in dodecane, and to the upper 96 well donor plate was added 200. mu.L of sample. Donor 96 well plates were placed on recipient 96 well plates to form a "sandwich" structure and allowed to stand at 25 ℃ for 10 h. The absorbance of the sample in the receptor plate was measured using a microplate reader (Infinite M200 Pro, TECAN, Salzburg, Austria). The permeability of CTFBA, EGCG, LK7 and BSA-B was calculated using the following formula:

in order to verify the detection result, 12 kinds of commercial drugs with known blood brain barrier permeability are introduced for linear correlation analysis, and the standard permeability limit capable of penetrating the blood brain barrier is obtained. Experimental measurements and theories in the literatureThe values are compared and the equation is obtained by linear fitting: p (exp) -1.04 p (lit) -0.03, (R)²0.96). According to the blood brain barrier permeability limit (P (lit) ═ 4.0X 10) given in the literature^-6cm/s) under the conditions of the experiment, if the permeability is higher than 4.1X 10^-6cm/s compounds are able to penetrate the blood-brain barrier with a permeability of less than 2.1X 10^-6cm/s of the compound is not able to cross the blood brain barrier.

As shown in FIG. 7, the P value of CTFBA is 11.96X 10^-6cm/s is stronger than a polypeptide inhibitor LK7 and a protein inhibitor BSA-B which do not have blood brain barrier penetrating ability, and is better than a small molecular inhibitor EGCG which also has the blood brain barrier penetrating ability, so that the CTFBA has good blood brain barrier permeability and has wide application prospect in AD treatment.

The invention provides a micromolecule inhibitor obtained by coupling glutamine on the surface of 3,4, 5-trihydroxybenzoic acid, and provides 4-carbamoyl-2- [ (3,4,5-trihydroxyphenyl) formamido]Application of butyric acid compound in preparation of drugs for inhibiting amyloid beta protein aggregation and application of butyric acid compound in A beta protein aggregation₄₂Aggregation, clearance, toxicity inhibition and blood brain barrier penetration experiments. Having described preferred embodiments in the field, it will be apparent to those skilled in the art that the techniques of the present invention may be practiced with modification, or with appropriate modification and combination, of the methods described herein without departing from the spirit, scope, and spirit of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and content of the invention.