Selective butyrylcholine esterase inhibitor and application thereof

Abstract

The invention discloses a carvacrol / thymol derivative with a structure as shown in the following formula (I), and pharmaceutically acceptable salts of the derivative: # imgabs0 #, wherein R is selected from 1-piperidyl or 1-pyrrolidinyl. The carvacrol / thymol derivative has remarkable inhibitory activity and high selectivity on butyrylcholine esterase, has good anti-neuroinflammation activity, can be used as a selective butyrylcholine esterase inhibitor with the anti-neuroinflammation activity, can be used for preparing medicines for preventing or treating Alzheimer's disease, and has wide application prospects. The compound has a good application prospect in treatment of Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to a butyrylcholinesterase inhibitor, in particular to a highly selective butyrylcholinesterase inhibitor. The butyrylcholinesterase inhibitor of the present invention also has anti-neuroinflammatory activity at the same time. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly, and its main clinical manifestations are memory loss and cognitive impairment. In the next few decades, with the increase in the aging population, AD will become one of the largest health burdens in the world.

[0003] The pathogenesis of AD is complex. Based on the cholinergic damage hypothesis, increasing the level of acetylcholine (ACh) in the brain and thus improving cholinergic nerve function is still the most important method in the current treatment of AD. In the initial stage of the disease, ACh is mainly hydrolyzed by acetylcholinesterase (AChE). As the disease progresses, the content and activity of AChE in the brains of AD patients significantly decrease, while the activity of butyrylcholinesterase (BuChE) increases and replaces AChE to play the role of hydrolyzing ACh. This also corroborates that clinically used AChE inhibitors such as donepezil, rivastigmine, and galantamine can only be mainly used to relieve the symptoms of mild or moderate Alzheimer's dementia.

[0004] In vivo experiments have proved that selective BuChE inhibitors can improve the memory and cognitive functions of AD model animals, and there are no peripheral nervous system side effects of AChE inhibitors. Therefore, the key to the treatment of severe Alzheimer's dementia lies in selectively inhibiting BuChE in the brain.

[0005] In addition, the "neuroinflammation hypothesis" has been gradually recognized by researchers in this field. Research from Nature Medicine found that amyloid plaques are present in the brains of many elderly people but never develop into Alzheimer's disease. The interaction between microglia-mediated neuroinflammation and amyloid proteins releases the propagation of tau protein, ultimately causing extensive brain damage and cognitive impairment. Intervening in neuroinflammation may help reverse or slow down the pathological accumulation of tau protein in the brain, thereby avoiding dementia, which has potential benefits for early Alzheimer's disease patients. Therefore, inhibiting neuroinflammation may become a new option for the treatment of mild or moderate Alzheimer's dementia.

[0006] Natural medicines, with their unique advantages and characteristics, provide more options for disease treatment. Carvacrol and thymol are isomers and belong to monoterpenoid compounds. They are commonly present in the volatile oils of various aromatic plants and have biological activities such as antibacterial, antioxidant, and anti-inflammatory effects. Structural modification and transformation of them can effectively improve the biological activities and selectivities of their derivatives, which is an important link in drug research and development.

[0007] Based on the structures of natural active carvacrol and thymol, derivatives with anti-neuroinflammatory activity and selective inhibitory activity against BuChE were designed, synthesized, and screened. This is more reasonable and valuable for research and application than intervening in only one pathological mechanism of Alzheimer's disease alone, and can provide more options for the prevention or treatment of Alzheimer's disease.

[0008] Kurt B Z, et al published research results on the synthesis and anti-cholinesterase activities of carvacrol and thymol derivatives (BIOORGAN MED CHEM, 2017, 25(4): 1352 - 1363). Among the carvacrol / thymol substituted carbamate derivatives involved, one hydrogen atom on the nitrogen atom of the carbamate moiety of the compound was substituted by a cyclopentyl, cyclohexyl, or aromatic group to form a secondary amine structure. Evaluation proved that this change significantly improved the selective inhibitory effect of the compound on butyrylcholinesterase. Among them, 34 compounds among the reported compounds had inhibitory activities against both AChE and BuChE. More specifically, for the selectivity index (Selectivity index, SI = AChE IC 50 / BuChEIC 50 ), among them, 14 had values less than or equal to 10, accounting for 41%; 11 had values greater than 10 and less than 100, accounting for 32%; 5 had values greater than 100 and less than 250, accounting for 15%; 2 had values greater than 250 and less than 700, accounting for 6%, and the SI values of the remaining 2 were up to 1130, and the corresponding inhibitory activity IC 50 value against butyrylcholinesterase was 0.13 ± 0.002 μM. SUMMARY OF THE INVENTION

[0009] Aiming at the problems existing in the prior art, the first object of the present invention is to provide a new carvacrol / thymol derivative for use in drugs for the prevention or treatment of Alzheimer's disease.

[0010] Providing the application of the carvacrol / thymol derivative as a selective butyrylcholinesterase inhibitor is another object of the present invention.

[0011] The third object of the present invention also lies in providing the application of the carvacrol / thymol derivative as a drug for inhibiting the inflammatory response activity of microglia.

[0012] To achieve the above-mentioned invention object, the present invention first screened and obtained a carvacrol / thymol derivative having the structure shown in the following formula (I), and a pharmaceutically acceptable salt of the derivative:

[0013] Wherein, R is selected from 1-piperidinyl or 1-pyrrolidinyl.

[0014] Furthermore, the carvacrol / thymol derivative of the present invention is selected from the following compounds, or a pharmaceutically acceptable salt thereof:

[0015] Piperidin-1-yl 5-isopropyl-2-methylphenyl ester, piperidin-1-yl 2-isopropyl-5-methylphenyl ester, pyrrolidin-1-yl 5-isopropyl-2-methylphenyl ester, pyrrolidin-1-yl 2-isopropyl-5-methylphenyl ester.

[0016] The "pharmaceutically acceptable salt" of the present invention refers to a conventional acid addition salt formed by retaining the biological activity and properties of the carvacrol / thymol derivative having the structure shown in formula (I) and combining with a suitable non-toxic inorganic acid or organic acid.

[0017] Furthermore, the carvacrol / thymol derivative having the structure shown in formula (I) of the present invention can be prepared by using carvacrol or thymol as a raw material and carrying out an esterification reaction with 1-piperidinecarbonyl chloride or pyrrolidine-1-carbonyl chloride.

[0018] More specifically, carvacrol or thymol is dissolved in an anhydrous CH 2 Cl 2 solvent system of 4-dimethylaminopyridine and N,N-diisopropylethylamine, and 1-piperidinecarbonyl chloride or pyrrolidine-1-carbonyl chloride is added at room temperature for esterification reaction.

[0019] Different from the method described in the literature of Kurt B Z, et al. where different substituted isocyanates are used as raw materials and react with phenolic hydroxyl groups under heating and reflux conditions, the present invention uses a formyl chloride substituted with a nitrogen heterocycle as a raw material and reacts with phenolic hydroxyl groups at room temperature. The yield of the carvacrol / thymol derivative is 85-94%, the reaction conditions are mild, and it is more suitable for industrial production.

[0020] It should be noted that the above preparation method is only a preferred preparation method of the derivative of the present invention, and is not used to limit the preparation method of the derivative of the present invention.

[0021] The carvacrol / thymol derivative with the structure shown in formula (I) of the present invention can be used to prepare a drug for preventing or treating Alzheimer's disease.

[0022] Taking donepezil as a positive control, the present invention detected the inhibitory activities of AChE and BuChE against the synthesized compounds, which showed high BuChE inhibitory activity at the nanomolar level, and at the same time had a low inhibitory activity against AChE, demonstrating a high selectivity for BuChE.

[0023] The research of the present invention also proves that the carvacrol / thymol derivative can inhibit the inflammatory response of microglia induced by LPS, showing good anti-neuroinflammatory activity.

[0024] Compared with the prior art, the carvacrol / thymol derivative of the present invention has significant inhibitory activity and high selectivity for butyrylcholinesterase, and has good anti-neuroinflammatory activity. It can be used as a selective butyrylcholinesterase inhibitor with anti-neuroinflammatory activity to prepare a drug for preventing or treating Alzheimer's disease and applied to the treatment of Alzheimer's disease, showing good application prospects.

[0025] Furthermore, the present invention also provides a drug for treating Alzheimer's disease, which contains the carvacrol / thymol derivative of the present invention and its pharmaceutically acceptable salts as drug active ingredients, and one or more pharmaceutically acceptable carriers or excipients.

[0026] The preparation process of the novel selective butyrylcholinesterase inhibitor with anti-neuroinflammatory activity provided by the present invention is simple and easy for industrial synthesis. Description of the Drawings

[0027] Figure 1 It is the Lineweaver-Burk plot of the interaction between compound SXF1 and butyrylcholinesterase.

[0028] Figure 2 It is the inhibition constant of compound SXF1 against butyrylcholinesterase ( K i ) plot.

[0029] Figure 3 It is the specific travel trajectory of mice in the water maze experiment. Embodiments

[0030] The following further describes the specific embodiments of the present invention in detail with reference to the drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, so that those skilled in the art can well understand and utilize the present invention, rather than limiting the protection scope of the present invention.

[0031] In the embodiments of the present invention, the production processes, experimental methods or detection methods involved, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps according to the names and apply the corresponding equipment, and implement them under conventional conditions or the conditions recommended by the manufacturer.

[0032] There are no special restrictions on the sources of various instruments, equipment, raw materials or reagents used in the embodiments of the present invention. They are all conventional products that can be obtained through regular commercial channels, and can also be prepared according to the conventional methods well-known to those skilled in the art.

[0033] The following embodiments of the present invention specifically provide a carvacrol / thymol derivative having the structure shown in the following formula (I) and its pharmaceutically acceptable salts:

[0034] Among them, R is selected from 1-piperidinyl or 1-pyrrolidinyl.

[0035] Specifically, the carvacrol / thymol derivative may be a compound with the structural formula shown in the following table.

[0036]

[0037] The "pharmaceutically acceptable salts" specifically refer to the conventional acid addition salts that can retain the biological activities and properties of the derivatives of formula (I) and are formed with suitable non-toxic inorganic acids or organic acids, and these salts are also included within the scope of the present invention. A list of suitable salts can be found in S. M. Brige et al., J. Pharm. Sci ., 1977, 66, 1-19.

[0038] The compounds involved in the embodiments of the present invention are compounds with a completely new structure that the inventors have creatively discovered based on the analysis of objective laws and specific practices, and have significant differences in structure, physical and chemical properties, and biological activities from the reported derivatives. The nitrogen atom in the carbamate moiety is a nitrogen-containing heterocycle, forming a tertiary amine structure, resulting in obvious changes in physical and chemical properties and certain differences in the binding characteristics with the target.

[0039] The compounds involved in the embodiments of the present invention have significant inhibitory effects and high selectivity on butyrylcholinesterase, and have good anti-neuroinflammatory activities, so they can be used in the preparation of drugs for preventing or treating Alzheimer's disease. Example

[0040] Example 1

[0041] 4 mL of 2-methyl-5-isopropylphenol (25.96 mmol), 0.317 g of 4-dimethylaminopyridine (2.596 mmol), 6.8 mL of N,N-diisopropylethylamine (38.94 mmol), and 20 mL of anhydrous CH 2 Cl 2 were successively added to a reaction flask as a solvent, and the mixture was stirred thoroughly for 10 min. Then, 4.8 mL of 1-piperidinecarbonyl chloride (38.94 mmol) was added, and the reaction was carried out at room temperature for 18 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporation, and the product was separated by silica gel column chromatography to obtain piperidine-1-carboxylic acid 5-isopropyl-2-methylphenyl ester as a pale yellow oily liquid with a yield of 94%, denoted as XQF1.

[0042] ESI-MS: m / z 262.85(M+1) + , C 16 H 23 NO 2 。

[0043] 1 H NMR (600 MHz, DMSO- d 6 ) δ 7.14 (d, J = 7.8 Hz, 1H), 6.99 (dd, J =7.8, 1.8 Hz, 1H), 6.88 (d, J = 1.8 Hz, 1H), 3.48 (d,4H), 2.83 (m,1H), 2.08(s, 3H), 1.60 (q, 6.2 Hz, 2H), 1.54 (d, Hz, 4H), 1.17 (d, 6H).

[0044] 13 C NMR (151 MHz, DMSO- d 6 ) δ 153.02, 150.20, 147.78, 130.91, 127.73,123.63, 120.55, 45.55, 45.07, 33.36, 26.08, 25.70, 24.24, 15.77.

[0045] Example 2

[0046] Using 5-methyl-2-isopropylphenol and 1-piperidinecarbonyl chloride as raw materials, under the same conditions as in Example 1, piperidine-1-carboxylic acid 2-isopropyl-5-methylphenyl ester was prepared as a pale yellow oily liquid with a yield of 93%, denoted as SXF1.

[0047] ESI-MS: m / z 262.82(M+1) + , C 16 H 23 NO 2 。

[0048] 1 H NMR (600 MHz, DMSO- d 6 ) δ 7.17 (d, J = 7.9 Hz, 1H), 6.98 (dd, J =8.0, 1.8 Hz, 1H), 6.81 (d, J = 1.8 Hz, 1H), 3.48 (d, 4H), 2.95 (m, 1H), 2.25(s, 3H), 1.62 – 1.58 (m, 2H), 1.53 (d, 4H), 1.14 (d, 6H)。

[0049] 13 C NMR (151 MHz, DMSO- d 6 ) δ 153.44, 148.92, 137.33, 136.24, 126.63,126.40, 123.70, 45.13, 27.22, 26.15, 25.73, 24.24, 23.19, 20.75。

[0050] The in vivo acute toxicity of compound SXF1 was tested. SPF-grade ICR mice (6 - 8 weeks old, 20 - 25 g) were purchased from Vital River Laboratories in Beijing. The animals and related disposals conformed to the requirements of animal welfare. The experiment had passed the ethical review of the Animal Welfare Ethics Committee of this institution before it was carried out.

[0051] Forty mice were randomly divided into a control group and compound SFX1 treatment groups (300, 400, 600, 800 mg / kg), with 8 mice in each group and an equal number of males and females. The compound was dissolved in a mixed solution of DMSO, Tween 80 and normal saline (5 / 5 / 90, v / v / v) and administered by single gavage. The number of dead mice within 24 h after administration was recorded. The results are shown in Table 1.

[0052]

[0053] All the mice that died within 24 hours showed obvious hind limb convulsions about 20 - 40 minutes after drug administration, and the convulsion time was about 3 hours. The autopsy results showed that there were no obvious abnormal pathological phenomena in the brains, hearts, livers, spleens, lungs, and kidneys of the mice in the SXF1 treatment group compared with the control group.

[0054] The median lethal dose (LD 50 ) of compound SXF1 was calculated by the weighted regression method (Bliss method) to be 465.7 mg / kg (the 95% confidence interval was 368.2 mg / kg - 584.6 mg / kg).

[0055] Example 3

[0056] Using 2 - methyl - 5 - isopropylphenol and pyrrolidine - 1 - carbonyl chloride as raw materials, with other conditions the same as in Example 1, a pale yellow oily liquid of 5 - isopropyl - 2 - methylphenyl pyrrolidine - 1 - carboxylate was prepared, with a yield of 90%, denoted as XQF2.

[0057] ESI - MS: m / z 248.86(M + 1) + , C 15 H 21 NO 2 .

[0058] 1 H NMR (600 MHz, DMSO - d 6 ) δ 7.14 (d, J = 7.6 Hz, 1H), 6.99 (dd, J =7.7, 1.9 Hz, 1H), 6.92 (d, J = 1.8 Hz, 1H), 3.51 (t, 2H), 3.35 – 3.32 (m,2H), 2.95 – 2.74 (m, 1H), 2.09 (s, 3H), 1.87 (dp, 6.7 Hz, 4H), 1.17 (d, 6H).

[0059] 13 C NMR (151 MHz, DMSO - d 6 ) δ 152.49, 150.13, 147.73, 130.90, 127.74,123.60, 120.55, 46.66, 46.46, 33.36, 25.83, 24.96, 24.25, 15.86.

[0060] Example 4

[0061] Using 5-methyl-2-isopropylphenol and pyrrolidine-1-carbonyl chloride as raw materials, with other conditions the same as in Example 1, a pale yellow oily liquid of 2-isopropyl-5-methylphenyl pyrrolidine-1-carboxylate was prepared, with a yield of 85%, denoted as SXF2.

[0062] ESI-MS: m / z 248.89(M+1) + , C 15 H 21 NO 2 。

[0063] 1 H NMR (600 MHz, DMSO- d 6 ) δ 7.17 (d, J = 7.9 Hz, 1H), 6.98 (dd, J =8.0, 1.8 Hz, 1H), 6.85 (d, J = 1.8 Hz, 1H), 3.50 (t, 2H), 3.35 – 3.32 (m,2H), 2.99 (m, 1H), 2.25 (s, 3H), 1.87 (dq, 4H), 1.14 (d, 6H).

[0064] 13 C NMR (151 MHz, DMSO- d 6 ) δ 152.87, 148.81, 137.35, 136.17, 126.57,126.39, 123.67, 46.68, 46.46, 27.15, 25.81, 24.94, 23.24, 20.78.

[0065] Comparative Example 1

[0066] According to the conditions in Example 1, N,N-dimethylcarbamoyl chloride and N,N-diethylcarbamoyl chloride were respectively reacted with 2-methyl-5-isopropylphenol to prepare 5-isopropyl-2-methylphenyl N,N-dimethylcarbamate and 5-isopropyl-2-methylphenyl N,N-diethylcarbamate, denoted as XQF-Me and XQF-Et respectively.

[0067] Comparative Example 2

[0068] According to the conditions in Example 1, N,N-dimethylcarbamyl chloride and N,N-diethylcarbamyl chloride were respectively reacted with 5-methyl-2-isopropylphenol to prepare 2-isopropyl-5-methylphenyl N,N-dimethylcarbamate and 2-isopropyl-5-methylphenyl N,N-diethylcarbamate, which were respectively denoted as SXF-Me and SXF-Et.

[0069] Comparative Example 3

[0070] According to the conditions in Example 1, N-methyl-N-ethylcarbamyl chloride was respectively reacted with 2-methyl-5-isopropylphenol and 5-methyl-2-isopropylphenol to prepare 5-isopropyl-2-methylphenyl N-ethyl-N-methylcarbamate and 2-isopropyl-5-methylphenyl N-ethyl-N-methylcarbamate, which were respectively denoted as XQF-MeEt and SXF-MeEt.

[0071] Example 5

[0072] Using the clinically used AChE inhibitor donepezil as the positive control, and natural carvacrol, thymol, and the 6 carvacrol / thymol derivatives synthesized in Comparative Examples 1-3 as the comparison, the acetylcholinesterase and butyrylcholinesterase inhibitory activities of the carvacrol / thymol derivatives synthesized in Examples 1-4 of the present invention were tested.

[0073] Add 100 μL of 5,5'-dithiobis(2-nitrobenzoic acid) to each well of a 96-well plate, then add 20 μL of different concentrations of the test compound or blank control to the corresponding wells. Add 50 μL of acetylcholinesterase or butyrylcholinesterase (0.22 U / mL) to the sample wells and standard control wells, mix well, and incubate at 37 °C for 10 min. Finally, add 10 μL of acetylthiocholine iodide or butyrylthiocholine iodide, mix well, and incubate at 37 °C for 15 min. The absorbance of the sample at 412 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the inhibition rate of each test compound against acetylcholinesterase or butyrylcholinesterase was calculated. All experiments were performed in triplicate and repeated independently 3 times.

[0074] Inhibition rate (%) = [1 - ( A 样品 - A 空白 ) / ( A 标准 - A 空白 )] × 100%

[0075] A 样品 is the absorbance value corresponding to the addition of the test compound, cholinesterase, substrate, and chromogenic agent; A 标准is the absorbance value corresponding to adding cholinesterase, substrate, and chromogenic reagent; A 空白 is the absorbance value corresponding to adding only substrate and chromogenic reagent.

[0076] The specific test results are shown in Table 2.

[0077]

[0078] In the above table, the unit of IC 50 is μM, representing the compound concentration corresponding to when the inhibition rate of the compound on acetyl / butyryl cholinesterase reaches 50%; IR represents the inhibition rate of 100 μM compound on acetyl / butyryl cholinesterase. If the inhibition rate does not exceed 50%, the IC 50 value will not be further measured; SI represents the ratio of the IC 50 of the compound inhibiting acetylcholinesterase to the IC 50 of inhibiting butyrylcholinesterase.

[0079] First, it can be seen from the results in Table 2 that except for SXF-Et, the rest of the derivatives all show good inhibitory activity against BuChE (derived from horse serum), but obviously the inhibitory activity of the compounds with the structure described in the present invention is better. Especially among them, XQF1, SXF1, and SXF2 even show nanomolar-level BuChE inhibitory activity, and the inhibitory activity of SXF1 against BuChE is the strongest (IC 50 =0.05 ± 0.003 μM), which is 148 times that of donepezil (IC 50 =7.42 ± 0.05 μM). Secondly, it is XQF1 (IC 50 =0.09 ± 0.003 μM), which is 82 times that of donepezil. It should be noted that the inhibition rates of 100 μM parent compounds carvacrol and thymol on BuChE are both lower than 20%.

[0080] Secondly, it can also be seen that the inhibitory activity of the compounds listed in Table 2 against AChE is low, and the inhibition rates at 100 μM are all less than 20%, far lower than that of donepezil (IC 50 =0.08 ± 0.002 μM).

[0081] Thus, it can be seen that the compounds with the structure described in the present invention have high selectivity for BuChE.

[0082] Comparative analysis shows that the SI values of the 6 alkyl nitrogen-substituted compounds in Comparative Examples 1-3 in Table 2 (except for SXF-Et) are 10, 28, 46, 60, and 73 in sequence, all of which are lower than 100. Among them, the inhibition rates of SXF-Et on BuChE and AChE are both lower than 50% at 100 μM, indicating relatively low cholinesterase inhibitory activity. The SI values of the 4 nitrogen heterocycle-substituted compounds in Examples 1-4 are 33, 385, 1111, and 2000 in sequence. Compared with the compounds in Comparative Examples 1-3, the SI values increase significantly (except for XQF2). In particular, the SI value of SXF1 is 2000, and the IC 50 value for inhibiting butyrylcholinesterase reaches 0.05 ± 0.003 μM, making it the most promising selective butyrylcholinesterase inhibitor.

[0083] Example 6

[0084] In the pathological process of AD, microglia promote the release of inflammatory factors by activating inflammasomes mainly composed of NLRP3. The NLRP3 inflammasome is composed of NLRP3 protein and ASC protein. Its activation promotes the cleavage of pro-caspase-1 into active caspase-1, triggering the maturation and release of downstream pro-inflammatory factor IL-1β, leading to neuroinflammatory responses and brain tissue damage.

[0085] Microglia can release IL1β under LPS induction and are often used as an in vitro neuroinflammation model. In this invention, the effects of the synthesized carvacrol / thymol derivatives in Examples 1-4 on LPS-induced microglial inflammatory responses were tested using this model.

[0086] The experimental mouse microglia (BV2) were provided by Shanghai Saibakang Biotechnology. First, the CCK8 method was used to detect the effects of various compounds at a concentration of 10 μM on the viability of BV2 cells.

[0087] BV2 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 2×10 4 / well and cultured overnight to allow the cells to adhere. In each sample group, 11 μL / well of the corresponding compound was added to make the final concentration 10 μM, and the cells were treated for 24 h. The Control group was added with an equal volume of solvent as a control.

[0088] The CCK8 detection results showed that there was no significant difference in the survival rate of BV2 cells between the compounds in each example at 10 μM and the Control group. The survival rate of XQF1 was 99.4%, the survival rate of XQF2 was 99.6%, the survival rate of SXF1 was 100.8%, and the survival rate of SXF2 was 100.9%. The concentration of 10 μM was safe for cell toxicity and could be selected as the initial concentration for subsequent experiments.

[0089] BV2 cells in logarithmic growth phase were counted, and the cell concentration was adjusted. Then, the cells were seeded into 96-well plates at a density of 2×10 4 / well and divided into a Control group, an LPS group, and a sample group. The cells were cultured overnight to allow them to adhere to the plate.

[0090] The Control group and the LPS group were added with 100 μL / well of complete medium containing DMSO, and the sample group was added with 100 μL / well of the corresponding compound solution, so that the final concentration was 10 μM for all groups. After 2 h of treatment, the Control group was added with 11 μL / well of blank medium, and the LPS group and the sample group were added with 11 μL / well of LPS solution at a concentration of 10 μg / mL and continued to be treated for 24 h. Each treatment group was repeated 3 times. The cell supernatant was taken and detected according to the instructions of the ELISA detection kit.

[0091] The experimental results are shown in Table 3.

[0092]

[0093] IR represents the inhibition rate of the compound on the release of IL1β at a concentration of 10 μM.

[0094] As can be seen from the results in Table 3, the inhibition rates of all compounds on the release of IL1β in LPS-induced BV2 cells at a concentration of 10 μM were greater than 50%, showing good anti-neuroinflammatory activity.

[0095] Example 7

[0096] For the thymol derivative SXF1 synthesized in Example 2, kinetic tests of butyrylcholinesterase were carried out to reveal the strong inhibitory effect of SXF1 on butyrylcholinesterase from the perspective of enzyme reaction kinetics.

[0097] 100 μL of 5,5'-dithiobis(2-nitrobenzoic acid) was added to each well of a 96-well plate, and then 20 μL of SXF1 at concentrations of 0.2 μM, 0.4 μM, 0.8 μM and a blank control without SXF1 were added to the corresponding wells. Subsequently, 50 μL of butyrylcholinesterase (0.22 U / mL) was added to the sample wells and the standard control wells. After mixing, the mixture was incubated at 37 °C for 10 min. Finally, 10 μL of substrates (Substrate) iodothiocholine at different concentrations was added to control the final concentrations to be 0.889 mM, 0.444 mM, 0.222 mM, 0.111 mM, 0.056 mM. After mixing, the mixture was incubated at 37 °C for 15 min, and the time scan curve of the compound at 412 nm was detected to measure the kinetics of the compound's inhibition of butyrylcholinesterase. All experiments were performed in 3 replicates and independently repeated 3 times.

[0098] By performing a linear regression on the enzyme-catalyzed reaction time and the absorbance value of the reaction product 2-nitro-5-thiobenzoate anion (TNB), the enzyme-catalyzed reaction rate can be obtained. V max (see Table 4).

[0099]

[0100] Then, by plotting 1 / [V] against 1 / [S], the Figure 1 Lineweaver-Burk plot of the compound against butyrylcholinesterase is obtained, and the slopes of the four straight lines are obtained. By plotting the slopes (see Figure 2 ), the absolute value of the intersection point of the straight line with the X-axis, 0.31, is the inhibition constant K i .

[0101] The study on the BuChE binding kinetics of the representative compound SXF1 showed that as the compound concentration increased, the maximum reaction rate of the enzyme ( V max) decreased, Km the K i value remained unchanged. Therefore, the compound was determined to be a non-competitive inhibitor of BuChE, and the inhibition constant

[0102] Example 8

[0103] Using donepezil as a control, the Morris water maze experiment was used to test the biochemical indexes of mice after administration of the thymol derivative SXF1 synthesized in Example 2, and to evaluate the cognitive and memory abilities of mice.

[0104] The test animals were male ICR mice (6 - 8 weeks old, 20 - 25 g), purchased from Vital River Laboratories (Beijing).

[0105] Forty-eight mice were randomly divided into 6 groups: blank group, model group, donepezil group, low-dose SXF1 group, medium-dose SXF1 group, and high-dose SXF1 group (n = 8).

[0106] The donepezil group was orally administered donepezil at 6 mg / kg, and the low-, medium-, and high-dose SXF1 groups were orally administered SXF1 at 5 mg / kg, 15 mg / kg, and 45 mg / kg, respectively. The blank group and the model group were orally administered an equal volume of normal saline.

[0107] The model group, the donepezil group, and the SXF1 group were intraperitoneally injected with scopolamine 1 h after oral administration of the drug, and the blank group was intraperitoneally injected with normal saline 1 h after oral administration of the solvent.

[0108] A 130-cm-diameter and 60-cm-high circular pool was filled with water, and an 8-cm-diameter escape platform was placed 1 cm below the water surface. The water temperature was maintained at 25 °C. Mice were placed on the escape platform for training on days 1 to 6 after administration of the drug. On day 7, the platform was removed, and the time and number of times the mice reached the position where the platform had been located were recorded.

[0109]

[0110] Figure 3 The specific travel trajectories of the mice in the water maze are shown. The experimental results are shown in Table 5. Compared with the blank group, the spatial learning and memory abilities of the mice in the model group were impaired, as manifested by a significant increase in the time to reach the platform ( P <0.05), a significant decrease in the number of times reaching the platform ( P <0.05), and a significant increase in the degree of chaos of the movement trajectories. Compared with the model group, the time for the mice in the medium- and high-dose SXF1 groups to reach the platform was significantly reduced ( P <0.05), comparable to that of donepezil, indicating that after treatment with SFX1, the ability of the mice to learn and remember the position of the platform was improved; compared with the model group, the number of times the mice in the high-dose SXF1 group passed through the original platform position was significantly increased ( P <0.05), better than that of donepezil, further indicating that after treatment with SFX1, the ability of the mice to learn and remember the position of the platform was improved, and the purposefulness of the movement trajectories was enhanced.

[0111] After the behavioral experiments were completed, the sera and brain tissues of the mice were collected. A mouse acetylcholine content detection kit and a mouse acetylcholinesterase / butyrylcholinesterase activity detection kit were used to detect the ACh content and AChE / BuChE activities in the hippocampus of the mice, and a mouse interleukin-1β detection kit was used to detect the IL-1β content in the sera of the mice.

[0112]

[0113] Studies have suggested that a decrease in the level of ACh in the brain leads to memory impairment. As shown in Table 6, the ACh level in the hippocampus of the mice in the model group was significantly decreased ( P <0.05), and a decrease in the learning and memory abilities of the mice in this group was also observed in the behavioral experiments. Compared with the model group, medium- and high-dose SXF1 could significantly up-regulate the ACh level ( P <0.05) and restore the learning and memory abilities of the mice.

[0114] AChE and BuChE are two key enzymes that hydrolyze ACh. The activities of the two enzymes in the mice in the model group were significantly increased ( P <0.05), resulting in increased hydrolysis of ACh and a decrease in the ACh level. Treatment with donepezil significantly decreased the AChE activity ( P<0.05), but does not affect the activity of BuChE. However, medium and high doses of SXF1 can significantly inhibit the activity of BuChE ( P <0.05), and has no effect on the activity of AChE, which is consistent with the results of in vitro enzyme activity screening, fully indicating that SXF1 increases the level of ACh mainly by inhibiting the activity of BuChE.

[0115] In addition, high-dose SXF1 can significantly reduce the level of IL1β and alleviate the inflammatory response, which is also consistent with the results of cell experiments. Thus, SXF1 is a highly selective butyrylcholinesterase inhibitor with anti-neuroinflammatory activity.

[0116] The above embodiments of the present invention do not elaborate all the details, nor limit the present invention to the above-described embodiments. Various changes, modifications, substitutions, and variations made to these embodiments by those of ordinary skill in the art without departing from the principle and purpose of the present invention shall be included within the protection scope of the present invention.

Claims

1. A carvacrol / thymol derivative, which is a compound having a structure represented by the following formula (I), and a pharmaceutically acceptable salt thereof: ， in, R is selected from 1-piperidinyl or 1-pyrrolidinyl.

2. The carvacrol / thymol derivative according to claim 1 is selected from the following compounds or pharmaceutically acceptable salts thereof: piperidine-1-carboxylic acid-5-isopropyl-2-methylphenyl ester, piperidine-1-carboxylic acid-2-isopropyl-5-methylphenyl ester, pyrrolidine-1-carboxylic acid-5-isopropyl-2-methylphenyl ester, pyrrolidine-1-carboxylic acid-2-isopropyl-5-methylphenyl ester.

3. The method for preparing the carvacrol / thymol derivative according to claim 1 is to use carvacrol or thymol as a raw material and carry out an esterification reaction with 1-piperidinyl chloride or pyrrolidine-1-carbonyl chloride.

4. The preparation method according to claim 3, characterized in that Carvacrol or thymol is dissolved in an anhydrous CH2Cl2 solvent system of 4-dimethylaminopyridine and N,N-diisopropylethylamine, and 1-piperidinyl chloride or pyrrolidine-1-carbonyl chloride is added at room temperature to carry out an esterification reaction.

5. Use of the carvacrol / thymol derivative according to claim 1 in the preparation of a drug for preventing or treating Alzheimer's disease.

6. Use of the carvacrol / thymol derivative according to claim 1 in the preparation of a highly selective butyrylcholinesterase inhibitor.

7. Use of the carvacrol / thymol derivative according to claim 1 in the preparation of a drug for inhibiting microglial inflammatory response.

8. Use of the carvacrol / thymol derivative according to claim 1 in the preparation of a selective butyrylcholinesterase inhibitor with anti-neuroinflammatory activity.

9. An anti-Alzheimer's disease drug, comprising the carvacrol / thymol derivative or a pharmaceutically acceptable salt thereof according to claim 1 as a pharmaceutically active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

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