Butyrylcholinesterase and monoamine oxidase B dual inhibitor and its application

Abstract

The present invention relates to the field of medical technology, and in particular to a compound of formula (I) which is a dual inhibitor of butyrylcholinesterase and monoamine oxidase B, and a preparation method and application thereof.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a butyrylcholinesterase and monoamine oxidase B dual inhibitor, a preparation method and application thereof. Technical Background

[0002] Neurodegenerative diseases are a significant health concern due to the aging population. Among people with neurodegenerative diseases, Alzheimer's disease accounts for the largest proportion, followed by Parkinson's disease. Alzheimer's disease is rapidly becoming an economic burden in various countries, and mortality associated with Alzheimer's disease is increasing. In addition, depressive symptoms are prevalent in individuals with neurodegenerative diseases, and according to a recent meta-analysis study, depression is present in approximately 40% of Alzheimer's disease cases and can be fatal. Summary of the invention

[0003] The present invention provides a dual inhibitor of butyrylcholinesterase and monoamine oxidase B, which has a structure as shown in the compound of formula (I). A compound of formula (I), its stereoisomers, tautomers, deuterated substances or pharmaceutically acceptable salts, has the following structure:

[0004]

[0005] In another aspect, the present invention provides a method for preparing a compound of formula (I), wherein the reaction process is as follows:

[0006]

[0007] A method for preparing a compound of formula (I), comprising the following steps:

[0008] 1) Synthesis of 4-methyl-1,3-benzo[d]thiazol-2-amine

[0009] Weigh o-toluidine and KSCN and add them to 1% glacial acetic acid, add liquid bromine to the glacial acetic acid to dilute it, slowly drop the glacial acetic acid solution of liquid bromine under ice bath conditions, remove the ice bath after the addition is complete, react at room temperature for 4 hours, pour the reaction solution into ice water, and adjust the pH to neutral with NaOH solution. After the solid precipitates, filter it with suction, dry the solid, and recrystallize it with ethanol to obtain 4-methyl-1,3-benzo[d]thiazole-2-amine;

[0010] 2) Synthesis of N-(Benzo[d]thiazol-2-yl)-2-chloroacetamide

[0011] 4-methyl-1,3-benzo[d]thiazol-2-amine was dissolved in tetrahydrofuran (THF), triethylamine was added, chloroacetyl chloride and THF were slowly added dropwise to the reaction flask under ice bath condition, after the addition was complete, the reaction was stirred, the solvent was evaporated under reduced pressure, the residue was washed with water to remove trimethylamine hydrochloride, the residue was dried, and the product N-(benzo[d]thiazol-2-yl)-2-chloroacetamide was obtained by recrystallization from EtOH;

[0012] 3) Synthesis of (R)-N-(Benzo[d]thiazol-2-yl)-2-(1-phenyl-3,4-dihydroisoquinolin-2(1H)-yl)acetamide

[0013] Dissolve N-(Benzo[d]thiazol-2-yl)-2-chloroacetamide in DMF, add triethylamine, and then slowly add (S)-1-phenyl-1,2,3,4-tetrahydroisoquinoline during stirring. Reflux in an oil bath at 80°C with stirring for 1.5-2 hours. Pour the reaction solution into ice water, adjust the pH to neutral with NaOH solution, filter and dry the precipitated solid, and recrystallize to obtain pure (R)-N-(benzo[d]thiazol-2-yl)-2-(1-phenyl-3,4-dihydroisoquinoline-2(1H)-yl)acetamide.

[0014] In yet another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), a stereoisomer, a tautomer, a deuterated substance or a pharmaceutically acceptable salt thereof.

[0015] The present invention provides a compound of formula (I), and use of its stereoisomer, tautomer, deuterated substance or pharmaceutically acceptable salt or its pharmaceutical composition in preparing medicines.

[0016] Furthermore, the prepared medicine is used in preparing medicine for treating and / or preventing neurodegenerative diseases.

[0017] Furthermore, the prepared medicine is used in preparing medicine for treating and / or preventing depression.

[0018] Furthermore, the prepared medicine is used in preparing medicine for treating and / or preventing neurodegenerative diseases and / or depression.

[0019] The compound of formula (I) of the present invention is a dual inhibitor of cholinesterase and monoamine oxidase B, and can be used as a drug for patients with comorbid neurodegenerative diseases and depression. Compared with other antidepressants, it can reduce side effects (such as hyponatremia, cardiac toxicity and increased bleeding); the compound of the present invention has multi-target effects, good drug efficacy and high selectivity.

[0020] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application, their stereoisomers, tautomers, deuterated substances or pharmaceutically acceptable salts and pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

[0021] The term "pharmaceutically acceptable excipients" refers to those excipients that have no significant irritation to the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0022] The pharmaceutical composition of the present invention can be prepared by combining the compound of the present application with suitable pharmaceutically acceptable excipients, for example, it can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

[0023] Typical routes of administration of the compounds of the invention or their pharmaceutically acceptable salts or their pharmaceutical compositions include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0024] The term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic in terms of partial or complete stabilization or cure of a disease and / or side effects resulting from a disease. As used herein, "treat" encompasses any treatment of a patient's disease that: (a) inhibits the symptoms of a disease, i.e., arrests its development; or (b) alleviates the symptoms of a disease, i.e., causes regression of the disease or symptoms.

[0025] The term "effective amount" means an amount of the compound of the present application that (i) treats or prevents a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. The amount of the compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the effect of the compound of formula (I) in Example 2 on the proliferation of L929 cells (MTT method).

[0027] Figure 2 This is the result of AO fluorescence staining experiment.

[0028] Among them, A is the effect of the blank group on the proliferation of L929 cells at 10X magnification (AO staining method); B is the effect of the blank group on the proliferation of L929 cells at 20X magnification (AO staining method); C is the effect of ID-01 on the proliferation of L929 cells at 10X magnification (AO staining method); D is the effect of ID-01 on the proliferation of L929 cells at 20X magnification (AO staining method). DETAILED DESCRIPTION

[0029] The following examples are used to further illustrate the present invention; however, they are not intended to limit or define the scope of the present invention.

[0030] MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;

[0031] 0.01M PBS buffer: weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4 and dissolve in 800mL distilled water, adjust the solution to 7.4 with HCl, and finally add distilled water to 1L;

[0032] MAO: monoamine oxidase;

[0033] MAO-B: monoamine oxidase B;

[0034] AChE: acetylcholinesterase;

[0035] BuChE: butyrylcholinesterase.

[0036] Example 1

[0037]

[0038] 1) Synthesis of 4-methyl-1,3-benzo[d]thiazol-2-amine

[0039] Weigh 50mmol o-Toluidine and 250mmol KSCN and join in the glacial acetic acid of about 15ml, 75mmol liquid bromine is joined in the glacial acetic acid of 2ml and diluted, under ice bath condition, slowly drip the glacial acetic acid solution of liquid bromine, after dropwise addition, remove ice bath, room temperature reaction 4h.Reaction solution is poured into ice water, pH is adjusted to neutrality with NaOH solution.After solid is separated out, suction filtration, solid drying, obtain 4-methyl-1,3-benzo[d]thiazole-2-amine with ethanol recrystallization.

[0040] 2) Synthesis of N-(Benzo[d]thiazol-2-yl)-2-chloroacetamide

[0041] 50mmol of 4-methyl-1,3-benzo[d]thiazol-2-amine was dissolved in tetrahydrofuran (THF), and 75mmol of triethylamine was added. Chloroacetyl chloride and THF (about 1.5ml) were slowly added dropwise to the reaction flask under ice bath conditions. After the addition was complete, the reaction was stirred for 1h, the solvent was evaporated under reduced pressure, and the residue was washed with water to remove trimethylamine hydrochloride. The residue was dried and recrystallized from EtOH to obtain the product N-(benzo[d]thiazol-2-yl)-2-chloroacetamide.

[0042] 3) Synthesis of (R)-N-(Benzo[d]thiazol-2-yl)-2-(1-phenyl-3,4-dihydroisoquinolin-2(1H)-yl)acetamide

[0043] Dissolve 50mmol N-(benzo[d]thiazol-2-yl)-2-chloroacetamide in DMF, add 75mmol triethylamine, slowly add 75mmol (S)-1-phenyl-1,2,3,4-tetrahydroisoquinoline during stirring, reflux in an oil bath at 80°C with stirring for 1.5-2 hours, pour the reaction solution into ice water, and adjust the pH to neutral with NaOH solution. After the solid precipitates, filter and dry it, and recrystallize to obtain pure (R)-N-(benzo[d]thiazol-2-yl)-2-(1-phenyl-3,4-dihydroisoquinoline-2(1H)-yl)acetamide.

[0044] Biological Research

[0045] Example 2

[0046] L929 cells in the logarithmic growth phase were selected, and the cell density was adjusted to 2×104 per well using a cell counter and inoculated into a 96-well plate. The 96-well plate inoculated with cells was placed in a 37°C 5% carbon dioxide cell culture incubator for 24 hours to allow it to grow completely attached to the wall. After 24 hours, culture media containing 100, 50, 25, 12.5, and 6.5 μM of the compound of formula (I) to be tested were added (a blank group was added with a medium without drug), and the cells were gently shaken and placed in a 37°C 5% carbon dioxide cell culture incubator for 24 hours. Each concentration was repeated 6 times. According to the classic MTT method, after 24 hours, add 20 μL of 5 mg / kg MTT solution (prepared in PBS with pH = 7.6) in the dark, and culture in the cell culture incubator for another 4 hours. After 4 hours, take out the 96 culture plate and discard the original culture medium, add 160 μL DMSO, put it on a shaker and mix it thoroughly for about 10 minutes, use a microplate reader to measure the absorbance at 490 nm, and record the results and analyze them.

[0047] The results of MTT assay are as follows Figure 1 As shown, compared with the blank control group, the test results show that at 0-100 μM, the survival rate of cells of the compound of formula (I) is more than 90%, and no obvious cytotoxicity is shown.

[0048] Example 3 Cell experiment - A0 staining analysis of cell viability

[0049] L929 cells were selected from the logarithmic growth phase and the cell density was adjusted to 8 × 10 per well using a cell counter. 5 Inoculate in a 6-well plate, place the 6-well plate inoculated with cells in a 37°C 5% carbon dioxide cell culture incubator and culture for 12 hours. After 12 hours, add 1 mL of culture medium containing 100 μM of the compound of formula (I) to be tested (a blank group is added with a medium without drugs), shake gently, and culture in a 37°C 5% carbon dioxide cell culture incubator for 12 hours. After 12 hours, discard the culture medium, add 0.01M PBS buffer to wash once, slowly add 1 mL 0.01M PBS buffer and 80 μL AO solution in a dark environment, mix for 5 minutes to make it evenly and fully stained, aspirate the stain after 5 minutes, and wash 1-2 times with 0.01M PBS buffer, observe under a fluorescence microscope, save the results and analyze.

[0050] The results of AO fluorescence staining experiments are shown in Figure 2 As shown, compared with the blank control group, the test results showed that at 100 μM, the compound of formula (I) showed no obvious cytotoxicity to L929 cells.

[0051] Example 3 Determination of butyrylcholinesterase and monoamine oxidase B inhibitory activity

[0052] Total MAO inhibitory activity

[0053] 25uL enzyme diluent (mother solution diluted 4 times), 25uL compound of formula (I) to be tested (reference group plus rasagiline, blank group plus 10% DMSO) were added to the 96-well ELISA plate in sequence, incubated at 37°C for 1h, then 120uL substrate was added, and then 80uL color development solution was added, incubated at 37°C for 30-60min, and the absorbance value was measured at a wavelength of 490nm using an ELISA reader. In addition, in order to exclude the influence of protein, a protein control group was set up, only the enzyme diluent was added, and the rest were replaced with 0.01M PBS buffer, and the absorbance value was measured at a wavelength of 490nm using an ELISA reader, and the results are shown in Table 1.

[0054] Table 1

[0055]

[0056]

[0057] MAO-B inhibitory activity and IC 50 Detection

[0058] 25uL enzyme solution and 25uL 10μM clorgyline solution were added to a 96-well plate in sequence, and after incubation at 37°C for 30min, 25uL of the compound of formula (I) to be tested (the control group was added with pargyline, and the blank group was added with 10% DMSO) was added, and incubated at 37°C for 1h, and then 120uL substrate was added, and then 80uL color development solution was added, and incubated at 37°C for 30-60min, and the absorbance value was measured at a wavelength of 490nm using an enzyme marker. In addition, in order to exclude the influence of protein, a protein control group was set up, and only the enzyme diluent was added, and the rest were replaced with 0.01M PBS buffer, and the absorbance value was measured at a wavelength of 490nm using an enzyme marker, and the results are shown in Table 2.

[0059] Table 2

[0060]

[0061] Cholinesterase inhibitory activity

[0062] The compound was dissolved in DMSO and then diluted in 10% DMSO to obtain sample solutions of different concentrations. Each concentration was tested three times in parallel, and 96 empty plates were taken, and 20 μL of the compound of formula (I) to be tested or the positive drug (wherein 10% DMSO was added to the blank control), 40 μL AChE or BuChE (0.2U / mL), 100 μL DTNB (0.001mol / L) were added in sequence, and incubated in a 37°C constant temperature water bath for 15 minutes after mixing evenly. Then, 20 μL of substrate ATCI or BTCI (0.001mol / L) was added, and after mixing evenly, the mixture was incubated in a 37°C constant temperature water bath for 3 minutes, and the absorbance value was measured at 412nm using a multifunctional microplate reader. The results are shown in Table 3.

[0063] Table 3

[0064]

[0065]

[0066] The test results of the inhibitory activity of the compound of formula (I) on MAO are shown in Table 1, the test results of the inhibitory activity of the compound of formula (I) on MAO-B are shown in Table 2, and the test results of the inhibitory activity of the compound of formula (I) on AChE and BuChE are shown in Table 3. The results show that the compound of formula (I) exhibits good inhibitory effects on MAO-B and BuChE.

[0067] Although the present invention has been comprehensively described through its embodiments, it is worth noting that various changes and modifications are obvious to those skilled in the art. Such changes and modifications should be included in the scope of the appended claims of the present invention.

Claims

1. A compound represented by formula (I), characterized in that: The structural formula is as follows:

2. A method for preparing a compound of formula (I) as claimed in claim 1, characterized in that: The reaction process is as follows:

3. A method for preparing a compound of formula (I) as claimed in claim 1, characterized in that: The following steps are involved: 1) Synthesis of 4-methyl-1,3-benzo[d]thiazol-2-amine Weigh o-toluidine and KSCN and add them to glacial acetic acid, add liquid bromine to the glacial acetic acid to dilute it, slowly drop the glacial acetic acid solution of liquid bromine under ice bath conditions, remove the ice bath after the dropwise addition is complete, react at room temperature for 4 hours, pour the reaction solution into ice water, adjust the pH to neutral with NaOH solution, filter after solid precipitation, dry the solid, and recrystallize it with ethanol to obtain 4-methyl-1,3-benzo[d]thiazole-2-amine; 2) Synthesis of N-(Benzo[d]thiazol-2-yl)-2-chloroacetamide Dissolve 4-methyl-1,3-benzo[d]thiazol-2-amine in tetrahydrofuran (THF), add triethylamine, slowly drop chloroacetyl chloride and THF into the reaction flask under ice bath conditions, stir the reaction after the addition is complete, evaporate the solvent under reduced pressure, wash the residue with water to remove trimethylamine hydrochloride, dry the residue, and recrystallize from EtOH to obtain the product N-(benzo[d]thiazol-2-yl)-2-chloroacetamide; 3) Synthesis of (R)-N-(Benzo[d]thiazol-2-yl)-2-(1-phenyl-3,4-dihydroisoquinolin-2(1H)-yl)acetamide Dissolve N-(Benzo[d]thiazol-2-yl)-2-chloroacetamide in DMF, add triethylamine, and then slowly add (S)-1-phenyl-1,2,3,4-tetrahydroisoquinoline during stirring. Reflux in an oil bath at 80°C with stirring for 1.5-2 hours. Pour the reaction solution into ice water, adjust the pH to neutral with NaOH solution, filter and dry the solid after precipitation, and recrystallize to obtain pure (R)-N-(benzo[d]thiazol-2-yl)-2-(1-phenyl-3,4-dihydroisoquinoline-2(1H)-yl)acetamide.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of the compound represented by formula (I) as claimed in claim 1.

5. Use of a compound of formula (I) as claimed in claim 1 or a pharmaceutical composition as claimed in claim 4 in the preparation of a drug.

6. The use according to claim 5, wherein the drug preparation is used in the preparation of drugs for treating and / or preventing neurodegenerative diseases.

7. The use according to claim 5, wherein the drug preparation is used in the preparation of drugs for treating and / or preventing depression.

8. The use according to claim 5, wherein the drug preparation is used in the preparation of drugs for treating and / or preventing neurodegenerative diseases and / or depression.

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