Application of small molecule compounds targeting Aβ oligomers in the preparation of drugs for alleviating or treating Alzheimer's disease

Abstract

The present invention discloses the use of a small molecule compound targeting Aβ oligomers in the preparation of a drug for alleviating or treating Alzheimer's disease, which can significantly improve the behavioral abnormalities of APP / PS1 mice. In particular, the compound PTO-29 can quickly penetrate the blood-brain barrier as a probe, and after binding to amyloid protein in the brain, it plays a good role in the early diagnosis of Alzheimer's disease; it has good safety after long-term administration, delays the progression of Alzheimer's disease and achieves a good therapeutic effect.

Description

Technical Field

[0001] The present invention relates to the use of a small molecule compound targeting Aβ oligomers in the preparation of a drug for alleviating or treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a fatal neurodegenerative disease caused by multiple factors, accounting for about 50-60% of dementia patients. The pathogenic factors of AD include the widely concerned pathological hypotheses, including the abnormal phosphorylation hypothesis of tau protein, the GSK-3β hypothesis, the metal homeostasis imbalance hypothesis, the cholinergic hypothesis, the neuroinflammation hypothesis, and so on. Due to the presence of Aβ plaques in the brain of patients, and the abnormal increase of Aβ production in people with APOEε4, APP, PSEN1, and PSEN2 gene mutations, the risk of AD is significantly increased. The Aβ cascade hypothesis has always been the focus of drug research. The amyloid precursor APP forms Aβ monomers under the dissociation of β-secretase and γ-secretase, mainly Aβ40 and Aβ42. Two subtypes: Aβ40 is a short peptide composed of 40 amino acids, and Aβ42 has two more amino acids at the C-terminus than Aβ40, namely Ile 41 and Ala 42, and the increased lipid solubility makes it easier to aggregate.

[0003] Under normal conditions, Aβ monomers maintain an irregular state and have no neurotoxicity. Under the induction of external environments such as pH and temperature, they undergo conformational transformation to form a β-sheet structure, which then aggregates through the hydrophobic region (Aβ16-22) to form dimers, trimers, hexamers, and dodecamers, which are highly neurotoxic soluble oligomers. The oligomers further polymerize into insoluble Aβ fibers that precipitate in the brain to form plaques. At present, there are four types of drugs approved for the treatment of AD in clinical practice: acetylcholinesterase inhibitors, N-methyl-D-aspartate inhibitors, low-molecular oligosaccharide compounds, and aducanumab, which acts on Aβ plaques in the brain. However, the first two types of drugs can only relieve AD symptoms, but cannot delay or prevent the progression of AD. The therapeutic effects of the latter two types of drugs are controversial and need to be further verified in clinical practice. With the increasing aging of society, there is a huge unmet clinical need for AD treatment drugs. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a small molecule compound targeting Aβ oligomers for use in the preparation of a drug for alleviating or treating Alzheimer's disease.

[0005] Technical solution: Use of the small molecule compound targeting Aβ oligomers described in the present invention in the preparation of drugs for alleviating or treating Alzheimer's disease.

[0006] Preferably, the small molecule compound targeting Aβ oligomers is as shown in Formula I:

[0007]

[0008] Wherein, R1 is -NRaRb, Ra and Rb are hydrogen, C1-6 alkyl or C1-6 substituted alkyl; Ra and Rb are not hydrogen at the same time

[0009] R2 is phenyl, C3-6 cycloalkyl or C1-4 substituted alkyl;

[0010] R3 is hydrogen, C1-6 alkyl or substituted alkane, halogen and alkoxy, R3 is monosubstituted or disubstituted;

[0011] X is a C atom or a N atom;

[0012] n=1, 2, 3, 4.

[0013] Preferably, C in R3 1-6 Alkyl is C 1-4 Alkyl, further, C 1-4 The alkyl group is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl; 3-6 The cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. Furthermore, R2 is preferably hydrogen, 3-ethyl or 2,5-dimethyl; R1 is N,N-dimethylamino, N,N-diethylamino or N,N-methylhydroxyethylamino.

[0014] Preferably, the compound described in the present application is selected from any of the following compounds:

[0015]

[0016] Preferably, the small molecule compound PTO-29 targeting Aβ oligomers shown in Formula V is:

[0017]

[0018] Among them, R1 is -NRaRb, Ra and Rb are both methyl, R2 is cyclopropyl, R3 is ethyl, and n=2.

[0019] Preferably, the dosage form of the drug is one of pills, ointments, tablets, oral liquids, subcutaneous injections and intravenous injections.

[0020] Preferably, the drug further comprises a stabilizer, a buffer, a solubilizer, an emulsifier, a diluent and / or an isotonic agent.

[0021] Preferably, the drug further comprises an excipient.

[0022] The inventors have found that soluble Aβ oligomers are stronger neurotoxic substances, which have been present in the brain for more than 10 years before the onset of disease symptoms, causing damage to neural tissue; and the level of Aβ oligomers isolated from the brain of AD patients is closely related to the progression of the disease and the severity of clinical symptoms. At present, a number of drugs that can reduce the production of soluble oligomers or directly act on soluble amyloid proteins significantly improve patient cognition, such as small molecules ALZ-801 and BAN2401. The AD drug targeted by Aβ oligomers shown in Formula I has the following characteristics: improving the binding of drugs to oligomers through the "V-type" structure, introducing blocking groups such as cyclopropyl or cyclobutyl to the exposed methyl groups to improve the stability of the drug, achieving specific binding to soluble Aβ oligomers by adjusting the size of the steric group, extending the number of double bonds in the side chain or replacing the side chain benzene ring with an electron-rich heterocycle to extend the emission wavelength, and adjusting the Log P value by changing the amino substitution group, improving the metabolic kinetic properties such as initial brain absorption and elution rate.

[0023] Further, PTO-29 of Formula V is preferably R based on Formula I 1-3 The introduced large steric hindrance group p-ethylphenyl can form hydrophobic contacts and π-π stacking interactions with the Phe19\Val36 residues on the surface of Aβ oligomers, thereby increasing the selectivity of the drug for oligomers; and the N,N-dimethylaniline part on the side chain can be embedded in the "hole" formed by the trimer, increasing the binding with Aβ oligomers, regulating the Log P value, improving the drug metabolic kinetics, and improving the bioavailability of the drug in vivo.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the small molecule compound targeting Aβ oligomers of formula I is used in drugs for alleviating or treating Alzheimer's disease, which can effectively diagnose, alleviate and treat Alzheimer's disease, especially PTO-29 of formula V can quickly penetrate the blood-brain barrier as a probe, and after binding to amyloid protein in the brain, it plays a good role in the early diagnosis of Alzheimer's disease; in addition, PTO-29 can be used as a small molecule drug for the treatment of Alzheimer's disease, has good safety after long-term administration, and can significantly improve the behavioral abnormalities of APP / PS1 mice, and reduce the content of amyloid protein in the brain, delay the progression of Alzheimer's disease and achieve good therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A reaction formula for preparing a small molecule compound targeting Aβ oligomers;

[0026] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of the small molecule compound targeting Aβ oligomers;

[0027] Figure 3 The carbon NMR spectrum of the small molecule compound targeting Aβ oligomers;

[0028] Figure 4 This is a graph showing the changes in the mean weight of mice over time, where WT represents the wild-type mouse group, AD represents the model animal APP / PS1 mouse group, and PTO-29 represents the model animal APP / PS1 mouse drug administration group;

[0029] Figure 5 After performing the Y-maze test for each group of mice, the order in which each group of mice entered different arms was recorded, and the average alternation rate value was obtained;

[0030] Figure 6 is the movement distance of each group of mice in the Y-maze experiment;

[0031] Figure 7 This is the route map of the Y-maze experiment for each representative mouse group;

[0032] Figure 8 After the water maze experiment was performed for each group of mice, the average time to find the platform during the training period of Day 1 to Day 5 was recorded, and the escape latency was calculated;

[0033] Fig. 9 The number of times mice in each group entered the target quadrant and crossed the platform during the Day 6 test of the water maze experiment;

[0034] Fig.10 This is the route map of the water maze experiment for each representative mouse group;

[0035] Fig.11 This is the semi-logarithmic curve of blood drug concentration-time after oral administration of PTO-29 in rats;

[0036] Fig.12 This is the semi-logarithmic curve of blood drug concentration-time after intravenous injection of PTO-29 in rats. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0038] Example 1

[0039] The preparation of the small molecule compound PTO-29 targeting Aβ oligomers of formula V comprises the following steps:

[0040] (1) Synthesis of 1-cyclopropyl-2-(3,5-dimethylphenyl)-1,3-butanedione

[0041]

[0042] Anhydrous K2CO3 (2.76g, 20mmol), L-proline (230mg, 2mmol), CuI (95mg, 0.5mmol) were added to a 100ml three-necked reaction bottle in sequence, and N2 was used for protection. 20mL DMSO, 1-cyclopropyl-1,3-butanedione (0.59ml, 5mmol), and 1-ethyl-4-iodobenzene (0.724ml, 5mmol) were added to the bottle through a syringe. The reaction temperature was 90°C, and the reaction was carried out for 18h. TLC monitoring (PE:EA = 20:1) was performed. Post-treatment: After the reaction solution was cooled to room temperature, it was filtered, and the filtrate was extracted with ethyl acetate (EA) (3×10ml). The organic layers were combined, washed with saturated brine (3×10ml), and dried over anhydrous Na2SO4. After concentration, column chromatography was performed, and the eluent was PE:EA = 70:1. The product was dried in vacuo to obtain 480mg of a yellow solid with a yield of 42%.

[0043] (2) 4-cyclopropyl-5-(4-ethylphenyl)-2,2-difluoro-6-methyl-2H-1λ 3 ,3,2λ 4 -Synthesis of dioxapurines

[0044]

[0045] The product of Example 1 (1) (352 mg, 1.72 mmol) and tributyl borate (0.93 ml, 3.45 mmol) were added to a 25 ml reaction bottle, sealed with a rubber stopper and plugged with a N2 balloon needle, and boron trifluoride etherate (0.93 ml, 3.45 mmol) was slowly added dropwise, stirred at room temperature for 5 h, and monitored by TLC (PE:EA=6:1). Post-treatment: water was slowly added to quench the boron trifluoride etherate, and a white solid was precipitated. Petroleum ether was continued to be added to precipitate more solids, and 350 mg of white solids were obtained by suction filtration, with a yield of 81%.

[0046] (3) Synthesis of PTO-29

[0047]

[0048] Using compound PTO-29-2 (130 mg, 0.46 mmol) and N, N-dimethylaminocinnamaldehyde (82 mg, 0.46 mmol) as raw materials, toluene (2.5 mL), acetic acid (5.5 μL, 0.093 mmol), and tetrahydroisoquinoline (8.0 μL, 0.062 mmol) were added to a 25 ml reaction bottle, stirred magnetically, reacted at 65 ° C for 5 h, and monitored by TLC (DCM). Post-treatment: The reaction solution was directly sanded, column chromatography (DCM), concentrated, and recrystallized from petroleum ether to obtain 128 mg of dark purple powder, with a yield of 64% and a melting point of 74-78 ° C.

[0049] Example 2

[0050] The small molecule compounds targeting Aβ oligomers are as follows:

[0051]

[0052] Table 1. Changes in fluorescence intensity after small molecule compounds targeting Aβ oligomers bind to three Aβ proteins

[0053]

[0054] λex is the maximum excitation wavelength of the compound, λem is the maximum emission wavelength of the compound; I is the maximum fluorescence intensity of the compound itself; Io is the fluorescence intensity of the compound and Aβ 42 The maximum fluorescence intensity after oligomer binding, the wavelength corresponding to this fluorescence intensity is λ; I is the compound's own fluorescence intensity at λ wavelength, Im is the compound and Aβ at λ wavelength 42 The fluorescence intensity of monomer binding, Ia is the fluorescence intensity of the compound and Aβ at wavelength λ 42 Fluorescence intensity after polymer (fiber) binding.

[0055] This series of compounds has the properties of fluorescent probes, that is, most of the compounds have weak fluorescence intensity, but the fluorescence intensity is enhanced after binding to Aβ protein. Therefore, the binding of the compound to the protein can be judged by the change in fluorescence intensity, and the selectivity of the compound for Aβ protein can be judged by the ratio of fluorescence intensity after binding to different proteins, thereby determining the expected therapeutic effect of the compound. The specific binding conditions are shown in Table 1; as shown in the table, Formula V has the strongest binding effect with Aβ oligomers, and I o / I m is 4.00, I o / I a is 3.70; after changing the side chain or core group and the length of the conjugated chain, the fluorescence intensity changes, and the preferred formulas 2 to 9 also show good binding abilities.

[0056] AD drugs targeting the Aβ oligomers shown in Formula I have the following characteristics: improving the binding of drugs to oligomers through the "V-type" structure, introducing blocking groups (cyclopropyl, cyclobutyl) into the exposed methyl groups to improve the stability of the drugs, achieving specific binding to soluble Aβ oligomers by adjusting the size of the steric groups, extending the number of double bonds in the side chain or replacing the side chain benzene ring with an electron-rich heterocycle to extend the emission wavelength, and adjusting the Log P value by changing the amino substitution group to improve the metabolic kinetic properties such as initial brain absorption and elution rate.

[0057] Example 3

[0058] Evaluation of small molecule compounds targeting Aβ oligomers for the treatment of Alzheimer's disease:

[0059] The present invention uses APP / PS1 mice as model animals, administers a small molecule compound PTO-29 targeting Aβ oligomers by oral gavage, measures behavioral indices (water maze, Y maze) and in vivo pharmacokinetic parameters, and evaluates the efficacy of the small molecule compound PTO-29 targeting Aβ oligomers in treating Alzheimer's disease.

[0060] The SPF-grade APP / PS1AD mice used in the experiment were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., and were male.

[0061] In the following examples, the dose of PTO-29 was 12 mg / kg mouse (oral administration).

[0062] 1. Experimental Animals

[0063] Twenty-eight male APP / PS1 mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. Before the experiment, the mice were placed in a quiet environment away from strong light for one week to adapt to the environment. The ratio of light to dark time was 1:1, the room temperature was controlled at 22-25°C, the humidity was 55%, and the mice were able to eat and drink freely.

[0064] 2. Experimental drugs and consumables

[0065]

[0066] 3. Experimental equipment and devices

[0067]

[0068]

[0069] EP tube, syringe, gavage needle, cotton swab, medical alcohol, ear tag.

[0070] 4. Experimental Methods

[0071] 4.1 Randomization

[0072] Fourteen cages were taken and numbered with markers, with 2 mice placed in each cage. 20 APP / PS1 mice were randomly numbered 1-10 in group A and 11-20 in group B, with the first cage numbered 1-2, the second cage numbered 3-4, and then the 10th cage numbered 19-20; 8 homologous wild-type (WT) mice were randomly numbered 21-28 in group C, the 11th cage numbered 21-22, and then the 14th cage numbered 27-28. Each mouse was punched at the edge of the ear and an ear tag was placed according to the number.

[0073] Mouse grouping and drug administration:

[0074]

[0075] 4.2 Preparation of drug solution

[0076] Each group began to receive drug intervention after 7 days of adaptation.

[0077] The preparation method of the drug solution is as follows:

[0078] PTO-29+5% DMSO+15% castor oil polyoxyethylene+80% PBS

[0079] Accurately weigh 3.6 mg of PTO-29 into a 4.5 mL EP tube, add 150 uL DMSO to completely dissolve the drug, then add 450 uL castor oil polyoxyethylene, vortex for 5 min, add 2400 uL PBS solution, vortex for 5 min, and prepare a drug solution with a total volume of 3 mL and a concentration of 1.2 mg / mL.

[0080] The drug-treated groups were all given the drug by oral gavage at 0.1 mL / 10 g for a total of 5 months.

[0081] The blank solution was prepared as follows:

[0082] 5% DMSO + 15% castor oil polyoxyethylene + 80% PBS.

[0083] The model group and the normal group were given an equal amount of blank solution by oral gavage at 0.1 mL / 10 g for a total of 5 months.

[0084] During the experiment, the diet, activity and fur condition of each group of mice were observed daily, and the weight of the mice was weighed and recorded before daily administration. The obtained data were then summarized to obtain the mouse weight change curve to evaluate the effect of the drug on each group of mice. After 5 months of administration, the weight records of each group of mice are shown in Table 2 and Figure 4 The results showed that during the 5-month drug administration, the weight change trend of mice in the normal group and the drug administration group was similar, and the weight was slightly lower than that of mice in the model group, with an average difference of 1 to 2 g, indicating that the drug had no obvious side effects on mice and had good safety.

[0085] Table 2: Average weight of mice in each group at different ages

[0086]

[0087] Conduct behavioral experiments on mice, including open field test, novel object recognition test, Y maze test, and water maze test.

[0088] Example 4

[0089] Behavioral experiment of mice - Y maze experiment:

[0090] The results of the Y maze test of mice in each group are shown in Table 3 and Figure 5 Figure 6 Figure 7 As shown. The results showed that the three groups of mice had similar average movement distances during the test, and on this basis, the order in which they entered the three maze arms was different. The higher the spontaneous alternation rate, the stronger the mouse's ability to distinguish different arms and the better its memory. The average alternation rates of mice in the normal group, model group, and drug-treated group were 73.4%, 70.0%, and 62.8%, respectively, indicating that APP / PS1 mice had cognitive defects, which was manifested as a decrease in the average alternation rate, while the cognitive function of APP / PS1 mice in the drug-treated group was significantly improved. In addition, the cognitive ability of APP / PS1 mice treated with PTO-29 was similar to that of wild-type mice.

[0091] Table 3: Y-maze test results

[0092]

[0093] Example 5

[0094] Behavioral experiment of mice - water maze experiment:

[0095] The results of the water maze test for each group of mice are shown in Table 4 and Table 5. Figure 8 Fig. 9 Fig.10 As shown. The escape latency refers to the time it takes for mice in each group to find the platform during the training period from Day 1 to Day 5. Result 1 shows that the time it takes for mice in the drug group and the normal group to find the platform gradually becomes faster during the training period, and during the last day of training, they can all find the platform within 35 seconds, and there is no significant difference between the two groups. However, the mice in the model group can find the platform after training, but it takes a long time. The average time to find the platform on the last day of training is 103s, which is significantly different from the first two groups. Result 2 shows that the behavioral data of mice in the Day 6 test period reflects the test of the mice's memory ability for the target platform during training, that is, the number of times the mice entered the target quadrant after the platform was removed, and the number of times they crossed the original platform. The results show that the mice in the model group showed obvious spatial memory impairment. The APP / PS1 mice treated with PTO-29 significantly improved the memory function of APP / PS1 mice and reversed the memory deficit of APP / PS1 mice.

[0096] Table 4: Water maze test results 1

[0097]

[0098] Table 5: Water maze test results 2

[0099]

[0100] Example 6

[0101] Plasma pharmacokinetic experiments of PTO-29, a small molecule compound targeting Aβ oligomers:

[0102] After the rats were given the drug PTO-29 by oral gavage and intravenous injection, the blood concentration of PTO-29 was observed over time, and the corresponding pharmacokinetic parameters and absolute bioavailability were calculated.

[0103] Six SD rats were divided into two groups and given PTO-29 by oral gavage and intravenous injection, respectively. About 0.25 mL of blood samples were collected from the jugular vein at 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, and 24h after intravenous injection and 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, and 24h after oral gavage. The concentration of PTO-29 in rat plasma samples was determined by LC-MS / MS, and the pharmacokinetic parameters were calculated using WinNolin software.

[0104] The pharmacokinetic experimental instruments and materials are as follows: SCIEX Triple Quad 5500+ triple quadrupole liquid chromatography-mass spectrometer, operating software is Analyst 1.7.2 (Applied Biosystems, Inc., USA); ExionLC liquid phase system; precision balance (Sartorius, model: GL124-1SCN); low-temperature high-speed refrigerated centrifuge (Eppendorf, model: 5424R); ultrapure water instrument (Thermo, model: MicroPure UV / UF); mixing oscillator (Qilin Bell, model: BE-3100). Methanol is chromatographically pure and purchased from Merck, Germany; acetonitrile is chromatographically pure and purchased from Merck, Germany; formic acid, purity ≥ 98%, purchased from Merck, Germany; dimethyl sulfoxide (DMSO) is chromatographically pure and purchased from J&K; EL, purchased from Wuhan New Earth Environmental Protection Materials Co., Ltd.; PBS, purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0105] Preparation method of dosing solution: accurately weigh 20.58 mg of the test sample PTO-29 and place it in a 15 mL EP tube, add 1.466 mL DMSO, 1.467 mL EL to dissolve, ultrasonicate and oscillate to mix until the reagent is purple-black and clear, then add 6.843 mL PBS, dissolve, ultrasonicate and oscillate to mix until the reagent is purple-black and clear, and prepare a 2 mg / mL test sample, and use this solution as a gavage and intravenous injection preparation. Prepare and use immediately.

[0106] This study adopted a single-dose single-cycle administration regimen as shown in Table 6. Six healthy and suitable animals were selected and divided into two groups, designated as Group 1 and Group 2.

[0107] Table 6: Dosing regimen for pharmacokinetic studies

[0108]

[0109] After administration, blood was collected from the jugular vein. The specific collection time points are shown in the following Table 7.

[0110] Table 7: Blood sample collection design

[0111]

[0112] The intravenous and oral administration doses of PTO-29 to rats were 4 mg / kg and 12 mg / kg, respectively. The blood concentration values ​​of PTO-29 after administration are shown in Tables 8-9.

[0113] Table 8: Pharmacokinetic parameters of PTO-29 in rats after intravenous injection (n=3)

[0114]

[0115] Table 9: Pharmacokinetic parameters of PTO-29 in rats after intragastric administration (n=3)

[0116]

[0117]

[0118] The changes of plasma drug concentrations over time in the two groups of rats after administration are as follows Fig.11 Fig.12 As shown, the results showed that PTO-29 could reach a higher blood concentration after tail vein injection and was slowly metabolized within 24 hours, indicating that the drug could exhibit better pharmacokinetic behavior and increase its brain exposure after injection.

[0119] It can be seen from the above results that the small molecule compound PTO-29 targeting Aβ oligomers of the present invention can significantly improve the behavioral abnormalities of APP / PS1 mice and the amyloid protein content in the brain, and delay the progression of AD. Therefore, the small molecule compound targeting Aβ oligomers of the present invention has good development prospects for the preparation of drugs for alleviating or treating AD.

Claims

1. Use of a small molecule compound targeting Aβ oligomers in the preparation of a drug for alleviating or treating Alzheimer's disease, characterized in that: The small molecule compound targeting Aβ oligomers is shown in Formula I: Wherein, R1 is -NRaRb, Ra and Rb are hydrogen, C1-6 alkyl or C1-6 hydroxyalkyl; R2 is phenyl, C3-6 cycloalkyl; R3 is hydrogen, C1-6 alkyl, halogen and alkoxy; X is a C atom or a N atom; n＝1、2、3、4。 2. Use of a small molecule compound targeting Aβ oligomers in the preparation of a drug for alleviating or treating Alzheimer's disease, characterized in that: The compound is selected from any one of the following compounds: 。 3. The use according to claim 1, characterized in that: The compound is PTO-29: 。 4. The use according to claim 1 or 2, characterized in that: The dosage form of the medicine is one of pills, ointments, tablets, oral liquids, subcutaneous injections and intravenous injections.

5. The use according to claim 1 or 2, characterized in that: The medicament may further include a buffer, a solubilizing agent, an emulsifier, a diluent and / or an isotonic agent.

6. The use according to claim 1 or 2, characterized in that: The medicament further comprises an excipient.

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