CN109535072B

CN109535072B - Acetylcholinesterase inhibitor and application thereof

Info

Publication number: CN109535072B
Application number: CN201811500165.XA
Authority: CN
Inventors: 张华�; 江成世; 朱孔凯; 张娟; 成志强; 宋佳丽; 葛永喜
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2018-12-10
Filing date: 2018-12-10
Publication date: 2020-09-01
Anticipated expiration: 2038-12-10
Also published as: CN109535072A

Abstract

The invention discloses an acetylcholinesterase inhibitor (A)E)‑1‑(3‑(4-ethylphenyl) -3-oxo-1-propenyl) -4- (pyrrole-1-yl) pyridine) and application thereof in preparing medicaments for preventing and/or treating AChE related diseases, A β aggregation related diseases and A β induced nerve injury caused diseases.

Description

Acetylcholinesterase inhibitor and application thereof

Technical Field

The invention relates to an acetylcholinesterase inhibitor and application thereof (E) -1- (3- (4-ethylphenyl) -3-oxo-1-propenyl) -4- (pyrrol-1-yl) pyridine) as acetylcholinesterase inhibitors.

Background

Alzheimer's Disease (AD) is a progressive, multifactorial, lethal neurodegenerative Disease. The clinical manifestations are cognitive disorder and abnormal behavior of patients. According to the worldwide report of Alzheimer's disease, the number of people suffering from AD in 2015 worldwide is four thousand six million, and the number of people suffering from AD in 2050 is expected to reach 1.3 hundred million.

The etiology and pathogenesis of AD is largely unknown, and choline dysfunction, amyloid- β (a β) aggregation, and tau protein phosphorylation are thought to play important roles in the development of AD. However, clinical trials targeting both a β and tau protein drugs have failed, and increasing cholinergic neurotransmission by increasing acetylcholine levels is still currently the most effective therapeutic approach for AD. Acetylcholine in the brain is predominantly hydrolyzed by acetylcholinesterase, and thus AChE inhibitors are the most studied and clinically most commonly accepted first-line drugs for AD, such as tacrine, donepezil, rivastigmine, and galantamine. However, these AChE inhibitors are not effective enough to provide limited relief or stabilization of the disease during specific stages of its development and are associated with severe side effects. Therefore, the development of novel AChE inhibitors with good therapeutic effects and low side effects is urgently needed.

In the process of new drug development, virtual screening is proved to be a very effective drug lead discovery means. The subject group carries out virtual screening on a commercial compound library (SPECS), three compounds with different structural types are obtained by active screening in the later period, and the compounds can inhibit acetylcholinesterase, can effectively inhibit A beta aggregation, have obvious neuroprotective effect on A beta induced nerve injury, can be used as a novel multifunctional acetylcholinesterase inhibitor, and have good application prospect in developing drugs for AD and other neurodegenerative diseases.

Disclosure of Invention

The invention aims to provide a novel multifunctional AChE inhibitor.

In a first aspect of the present invention, there is provided a compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof:

the compound is: (E) -1- (3- (4-ethylphenyl) -3-oxo-1-propenyl) -4- (pyrrol-1-yl) pyridine),

。

in a second aspect of the present invention, a pharmaceutical composition comprises a compound represented by formula I as described in the first aspect, or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; and a pharmaceutically acceptable carrier.

In a third aspect of the present invention, there is provided a compound of formula I as described in the first aspect, or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, for use in:

(i) preparing an AChE inhibitor;

(ii) preparing a medicament for preventing and/or treating AChE related diseases;

(iii) preparing an Abeta aggregation medicament;

(iv) preparing a medicament for preventing and/or treating diseases related to A beta aggregation;

(v) preparing a medicament with an obvious neuroprotective effect on A beta-induced nerve injury;

(vi) preparing a medicament for preventing and/or treating diseases caused by A beta-induced nerve injury.

Pharmaceutically acceptable carriers must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof, and generally suitable carriers, diluents and excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like. The particular carrier, diluent or excipient employed will depend upon the mode and purpose of administration of the compounds of the invention. The solvent is generally selected based on the solvents (GRAS) that one of skill in the art would consider safe for administration to mammals. Generally, safe solvents are non-toxic aqueous solvents (such as water) and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG400 or PEG300), and the like, and mixtures thereof. One or more buffering agents, stabilizing agents, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, slip agents, processing aids, colorants, sweeteners, flavorants, flavoring agents and other known additives that provide a tailored appearance to the drug (i.e., a compound of the invention or a pharmaceutical composition thereof) or aid in the manufacture of the drug product (i.e., for use in the preparation of a medicament) may also be included.

Advantageous effects

The compound can inhibit AChE, effectively inhibit A beta aggregation, has obvious neuroprotective effect on A beta induced nerve injury, and can be used for preparing medicines for preventing and/or treating AD and other related neurodegenerative diseases.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

The inventor of the application discovers a multifunctional AChE inhibitor through virtual screening and activity research of a commercial compound library, can effectively inhibit AChE, can effectively inhibit A beta aggregation, and has an obvious neuroprotective effect on A beta-induced nerve injury. On the basis of this, the present invention has been completed.

In the present invention, the compound of formula I refers to a compound having the following formula I:

。

EXAMPLE 1 Effect of Compounds on AChE enzymatic Activity

Acetylcholinesterase (AChE), thioacetylcholine iodide (ATC) as substrate and 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) as colour developer were purchased from Sigma. AChE inhibitory activity assays were performed with reference to the method reported by Ellan et al (Ellman, g.l. et al.Biochem. Pharmacol.1961, 7, 88.). mu.L of phosphate buffer (pH = 8.0) was added to each well of the 96-well plate, then 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 and 100. mu.M of 10. mu.L of test compound solution or blank was added to the corresponding air, followed by 10. mu.L of AChE, which was incubated for 5 min at 37 ℃ on a shaker. Adding 20 mu L of DTNB solution, placing the DTNB solution in a 37 ℃ shaking table for incubation for 5 min, then adding 10 mu L of substrate ATC, placing the substrate ATC in a 37 ℃ shaking table for incubation for 3 min, measuring the absorbance at 412 nm by using a microplate reader, and calculating the inhibition rate of the compound to be detected on AChE. Determination of the IC of the Compounds from the inhibition curves₅₀The values (inhibitor concentration at which the enzyme activity was inhibited by 50%) and the results are shown in Table 1.

TABLE 1 inhibitory Activity of Compounds on AChE enzymatic Activity

EXAMPLE 2 Effect of Compounds on A β aggregation

Mixing A β with DMSO (Sigma)_1-42(Gielsen)Physico-chemical Co., Ltd., Shanghai, China) was dissolved to prepare a stock solution of 200. mu.M.the stock solution was centrifuged at 13,500 rpm for 10 minutes to take the supernatant of the stock solution for experiment. the test compound was dissolved in DMSO to 0.8 mM. to indirectly test the ability of the compound to inhibit A β aggregation by measuring the fluorescence emission of ThT.76. mu.L of phosphate buffered saline (PBS of pH 7.4) was added to a 96-well plate, and 2. mu.L of 0.8 mM compound and 2. mu.L of 200. mu. M A β were added thereto_1-42After 24 hours of incubation at room temperature, 80. mu.L of 5. mu.M THT solution (dissolving THT in 50 mM glycine-NaOH buffer pH 8.5) was added to the reaction solution, fluorescence emission was measured at 490 nm in a microplate reader, excitation wavelength was 390 nm, the same spectrum was recorded by conducting three independent experiments, comparing fluorescence intensity, and% inhibition was calculated by the following equation 100- [ (Fi-Fb)/(Fo-Fb) × 100]Wherein Fi, Fo and Fb refer to inhibitor, THT and A β_1-42The fluorescence intensity obtained by the aggregation of A β in the presence of A β_1-42And the fluorescence intensity obtained in the presence of THT but in the absence of inhibitor; blank groups contained only THT. Donepezil was the positive control and galantamine was the negative control. The results of the experiment are shown in table 2.

TABLE 2 inhibitory Activity of Compounds on A.beta.aggregation

The experimental result shows that the compound I is opposite to the A β_1-42The aggregation has obvious inhibitory activity, and the action effect is better than that of a positive control medicament, namely donepezil.

EXAMPLE 3 Compound pair A β_1-42Protective effect for inducing SH-SY5Y nerve cell damage

SH-SY5Y was purchased from American national cell Bank, and cultured in DMEM medium containing 10% fetal bovine serum at 37 deg.C under 5% CO2 saturation humidity in an incubator at 2-2.5 × 10⁵The cells were seeded in 96-well plates at a density of one cell/mL, and experiments were performed after 24h of culture by changing fresh culture medium and dividing the cells into normal groups, A β_1-42Damage model group and test compoundAdding compounds I, II and III (10 mu M) into the treatment group of the compound to be detected, adding EGCG (tea polyphenol) into the EGCG positive control group, not adding the A β group and the normal group, adding A β with the final concentration of 10 mu M into each group except the normal group after culturing for 2 h_1-42The incubation was continued for 24 hours, MTT (final concentration 0.5 mg/ml) was added, and after 3 hours of incubation, the change in absorbance at 490 nm was measured. The results of the experiment are shown in table 3.

TABLE 3 Compound Pair A β_1-42Protective effect for inducing SH-SY5Y nerve cell damage

The experimental result shows that the compound I is opposite to the A β_1-42The induced SH-SY5Y nerve cell injury has obvious protective effect, and the effect is better than that of the positive control drug EGCG.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. The application of a pharmaceutical composition containing the compound shown in the formula I or the pharmaceutically acceptable salt thereof and the pharmaceutically acceptable carrier in preparing a medicament for preventing and/or treating AChE related diseases;

。

2. the use according to claim 1, wherein the agent for preventing and/or treating AChE-associated diseases is an acetylcholinesterase inhibitor.

3. The application of a pharmaceutical composition containing a compound shown as a formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier in preparing a medicament for preventing and/or treating diseases related to A beta aggregation;

。

4. the use according to claim 3, wherein the agent for preventing and/or treating a disease associated with A β aggregation is an agent for inhibiting amyloid- β aggregation.

5. The application of a pharmaceutical composition containing a compound shown as a formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier in preparing a medicament for preventing and/or treating diseases caused by A beta-induced nerve injury;

。

6. the use according to claim 5, wherein the medicament for the preparation of a medicament for the prophylaxis and/or treatment of a disease caused by a β -induced nerve damage is a medicament having a neuroprotective effect on a β -induced nerve damage.

7. The application of a pharmaceutical composition containing the compound shown in the formula I or the pharmaceutically acceptable salt thereof and the pharmaceutically acceptable carrier in preparing a medicament for preventing and/or treating AD-related neurodegenerative diseases;

。