CN110577480B - Preparation method and application of compound with anti-beta amyloid activity - Google Patents

Preparation method and application of compound with anti-beta amyloid activity Download PDF

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CN110577480B
CN110577480B CN201910641892.6A CN201910641892A CN110577480B CN 110577480 B CN110577480 B CN 110577480B CN 201910641892 A CN201910641892 A CN 201910641892A CN 110577480 B CN110577480 B CN 110577480B
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刘沙
周毅
张王刚
姜浩
李淋淋
郭婷婷
杨海泉
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Chengdu Medical College
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    • A61P25/00—Drugs for disorders of the nervous system
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Abstract

The invention provides a compound shown as a formula I or a pharmaceutically acceptable salt thereof. The invention also provides a preparation method of the compound. Experimental results show that the compound provided by the invention can obviously inhibit A beta-induced cytotoxicity, can obviously reduce the A beta level of an APP/PS1 transgenic mouse suffering from AD, improves the learning and memory capacity of the APP/PS1 transgenic mouse suffering from AD, and has good application prospects in preparation of beta-amyloid inhibitors and medicaments for preventing or treating Alzheimer's disease.
Figure DDA0002132164050000011

Description

Preparation method and application of compound with anti-beta amyloid activity
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a preparation method and application of a compound with anti-beta-amyloid activity.
Background
Alzheimer's Disease (AD) is the most common form of dementia (loss of memory) in older people. The major pathological changes in AD include extracellular accumulation of amyloid-beta (Α β) in the form of plaques and vascular disease and intracellular neurofibrillary tangles of aggregated hyperphosphorylated tau protein. Thus, lowering amyloid beta levels is an effective therapeutic strategy for the treatment of AD.
Beta amyloid is composed primarily of 39 to 42 amino acid peptides, produced by the sequential action of the proteases beta and gamma secretase, a larger precursor protein, called the Amyloid Precursor Protein (APP). Early onset of AD is due to genetic mutations in APP resulting in the overproduction of total beta amyloid protein, or its greater aggregation propensity 42 amino acids with type I. Furthermore, people with Down syndrome also have an extra chromosome, also containing the gene encoding APP, and therefore, elevated levels of β -amyloid protein are likely to develop AD.
Therefore, the research on the compounds with anti-beta amyloid activity has very important significance for preventing and treating Alzheimer's disease.
Research shows that arachidonic Acid Ethanolamine (AEA) is an endogenous cannabinoids substance, can be combined with cannabinoid receptors (CBR), capsaicin receptors (TR-PV1) and other non-cannabinoid receptors, regulates a nerve regulation mechanism, further regulates the cardiovascular system and plays a role in neuroprotection. However, AEA acts on multiple targets, which easily causes side reactions such as weight gain, fatigue, lethargy and the like; moreover, because of its short half-life, it is metabolized more rapidly in vivo and cannot maintain its basic activity, which brings difficulties to clinical application of AEA. The application of AEA and the analogues thereof in the anti-beta amyloid activity is not reported.
Therefore, the synthesis of novel compounds capable of targeting and resisting beta amyloid activity has very important opinions on the prevention and treatment of neurodegenerative diseases.
Disclosure of Invention
The invention aims to provide a compound with novel structure and anti-beta-amyloid activity, and a preparation method and application thereof.
The invention provides a compound shown as a formula I or a pharmaceutically acceptable salt thereof:
Figure BDA0002132164030000011
wherein R is1Selected from carboxyl, halogen, cyano, hydroxyl, C substituted by 0-3 substituents1-6Alkyl, C substituted by 0 to 3 substituents1-6Alkoxy, aryl substituted by 0-3 substituents, cycloalkyl substituted by 0-3 substituents, and heterocyclic group substituted by 0-3 substituents, wherein the substituents are independently selected from hydroxyl, -SH, and C1-6Alkyl radical, C2-6Alkenyl radical, C2-6Alkynyl, carboxyl, halogen, with 0-3R3Substituted aryl, substituted by 0-3R3Substituted azacyclic, guanidino, R4NHR5-、R4CONHR5-、R4NHCOR5-、R4SR5-，R3Selected from hydroxy, carboxy, halogen, C1-6Alkyl radical, C1-6An alkoxy group; r5Selected from none or C1-5Alkylene of (A), R4Is selected from H or C1-6An alkyl group;
R2is selected from C11-25Aliphatic hydrocarbon group of
Further, the air conditioner is provided with a fan,
R1selected from carboxyl, halogen, cyano, hydroxyl, C substituted by 0-3 substituents1-6Alkyl, each of said substituents being independently selected from hydroxy, -SH, C1-6Alkyl radical, C2-6Alkenyl radical, C2-6Alkynyl group, with 0 to 3R3Substituted phenyl, imidazolyl, tetrahydropyrrolyl, indolyl, guanidino, amino, R3Selected from hydroxyl, carboxyl, halogen;
R2is selected from C15-19And a linear hydrocarbon group containing 0 to 4 unsaturated double bonds, preferably C17And a straight chain hydrocarbon group having 1 to 4 unsaturated double bonds.
Further, the air conditioner is provided with a fan,
the structure of the compound is shown as formula II:
Figure BDA0002132164030000021
wherein R is1Selected from carboxyl, halogen, cyano, hydroxyl, C substituted by 0-3 substituents1-6Alkyl, each of said substituents being independently selected from hydroxy, -SH, C1-6Alkyl radical, C2-6Alkenyl radical, C2-6Alkynyl group, with 0 to 3R3Substituted phenyl, imidazolyl, tetrahydropyrrolyl, indolyl, guanidino, amino, R3Selected from hydroxyl, carboxyl and halogen.
Further, the air conditioner is provided with a fan,
wherein R is1Is selected from-CH2OH、
Figure BDA0002132164030000022
Carboxy, -CH2SH、
Figure BDA0002132164030000023
Figure BDA0002132164030000024
Further, the air conditioner is provided with a fan,
the compound is selected from the following structures:
Figure BDA0002132164030000025
Figure BDA0002132164030000031
the present invention also provides a process for preparing the above compound, comprising the steps of:
(1) n-hydroxysuccinimide with R2Reacting with-COOH to obtain a compound shown as a formula IV;
(2) and (3) reacting the compound shown in the formula III with the compound shown in the formula IV under an alkaline condition to obtain the compound.
Wherein the compound shown in the formula III is
Figure BDA0002132164030000032
The compound of formula IV is
Figure BDA0002132164030000033
Wherein R is1、R2As described above.
Further, in the step (1), the reaction is carried out under the action of a condensing agent, the reaction condition is room temperature and is protected from light, and the reaction time is 48-72 hours; preferably, the condensing agent is dicyclohexylcarbodiimide;
in the step (2), the alkaline condition is formed by adding organic alkali, the reaction temperature is room temperature, and the reaction time is 48-72 hours; preferably, the organic base is triethylamine.
The invention also provides the use of the above compound, or a pharmaceutically acceptable salt thereof, in the preparation of an amyloid beta inhibitor, preferably, the anti-amyloid beta inhibitor is a medicament for preventing or treating a neurodegenerative disease, more preferably, the neurodegenerative disease is alzheimer's disease.
The invention also provides a pharmaceutical composition which is prepared by taking the compound or the pharmaceutically acceptable salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
Further, the dosage form of the pharmaceutical composition is selected from powder, granules, capsules, solutions, emulsions or suspensions.
Experimental results show that the compound with a novel structure shown in the formula I can obviously inhibit A beta induced cytotoxicity, can obviously reduce the A beta level of an APP/PS1 transgenic mouse with AD, improves the learning and memory capacity of the APP/PS1 transgenic mouse with AD, and has a good application prospect in preparation of a beta amyloid inhibitor and a medicament for preventing or treating Alzheimer's disease.
The pharmaceutically acceptable salt of the invention is a product obtained by directly salifying the free base of the compound and an inorganic or organic acid. Wherein the inorganic or organic acid is selected from hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, picric acid, citric acid, maleic acid, methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, etc.
In the present invention, "substituted" means that 1, 2 or more hydrogen atoms in a molecule are replaced by other different atoms or molecules, including 1, 2 or more substitutions on the same atom or different atoms in the molecule.
In the present invention, Ca-bRefers to all groups or molecules having a-b carbon atoms; e.g. C1The-6 alkyl group means a straight or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl and the like.
As used herein, "aryl" refers to an all-carbon monocyclic or fused polycyclic group having a conjugated pi-electron system, such as phenyl and naphthyl. The aryl group may be fused to other cyclic structures (including saturated, unsaturated rings) but must not contain heteroatoms such as nitrogen, oxygen or sulfur, and the point of attachment to the parent must be at a carbon atom on the ring with the conjugated pi-electron system.
The aliphatic hydrocarbon group means a group remaining after an aliphatic hydrocarbon has lost one hydrogen atom, and includes a linear or branched, saturated or unsaturated hydrocarbon group.
Containing 0 to 4 unsaturationsThe term "double-bonded linear hydrocarbon group" means that the linear hydrocarbon group contains 0 to 4 double bonds
Figure BDA0002132164030000041
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
Figure 1 is a graph of the effect of a β on PC12 cell viability compared to normal groups:*P＜0.05，**P＜0.01，***P＜0.001 。
figure 2 is the effect of NITyr on PC12 cell viability compared to the a β group:*P＜0.05，**P＜0.01。
figure 3 is a graph of the effect of autophagy inhibitor 3MA on reversing NITyr on cell viability compared to a β group:*P＜0.05，**p is less than 0.01; comparison with NITyr group#P＜0.05。
FIG. 4 is a graph of the effect of NITyr on the ability of APP/PS1 transgenic mice to autonomously function, compared to APP/PS1 group:*P＜0.05。
FIG. 5 is a graph of the effect of NITyr on the locomotor ability of APP/PS1 transgenic mice, compared to APP/PS1 group:*P＜0.05，**P＜0.01，***P＜0.001。
FIG. 6 is the effect of NITyr on the learning and memory ability of APP/PS1 transgene, A: positioning navigation experiment; b: a space exploration experiment; compared with the APP/PS1 group,*P＜0.05，**P＜0.01，***P＜0.001。
figure 7 is a fluorescence plot of the effect of NIS on a β -induced PC12 cell damage.
FIG. 8 is NIS vs. AEffect of Α β levels in brain tissue of PP/PS1 transgenic mice, compared to APP/PS1 group:*P＜ 0.05，**P＜0.01，***P＜0.001。
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
The compound shown in the formula I is synthesized by the following synthesis method, which comprises the following steps:
(1) (1) N-hydroxysuccinimide with R2Stirring at 25 deg.C under the action of DCC in dark for 48 hr, vacuum filtering, extracting with water twice, and concentrating to obtain compound shown in formula IV; r2As described above.
(2) Reacting the compound shown in the formula III with the compound shown in the formula IV under the action of organic base, and obtaining the target compound of the invention through acidification and extraction.
R1、R2As described above.
Below is R2R in-COOH2Is composed of
Figure BDA0002132164030000051
The synthetic route of (1):
Figure BDA0002132164030000052
EXAMPLE 1 preparation of Compounds of the invention
(1) Preparation of intermediate A1
Adding 2.20mL (7.06mmol) of linoleic acid, 0.824g (7.16mmol) of N-hydroxysuccinimide and 40mL of ethyl acetate into a 100mL round-bottom flask, stirring at room temperature for dissolving, then gradually adding 1.468g (7.12mmol) of dicyclohexylcarbodiimide while stirring, stirring at 25 ℃ in the dark for 48 hours, pointing and tracking the reaction progress until the reaction of the linoleic acid is complete, filtering to remove a white precipitate DCU, adding water into filtrate for extracting twice, collecting and merging organic phases, and rotating under reduced pressure to obtain a yellow oily substance, namely the required intermediate A1 for later use.
(2) Linoleoylserine preparation (Compound 15, named NIS)
Figure BDA0002132164030000061
Dissolving the obtained intermediate A1 in 45mL 65% ethanol, adding 3mL (21.52mmol) triethylamine, adding 0.896g (8.53mmol) serine, and stirring at 25 deg.C in the dark for 72 h; after the reaction, the mixture was evaporated under reduced pressure to remove a large amount of ethanol, an appropriate amount of distilled water was added to the concentrate, pH was adjusted to 2 with concentrated HCl, an appropriate amount of ethyl acetate was added thereto, and the upper ethyl acetate layer was taken out, dried over anhydrous sodium sulfate, and concentrated. Silica gel column chromatography with dichloromethane/methanol (10/1) as mobile phase gave 1.30g of product as a pale yellow oil with 50.60% yield.
1H NMR(400MHz,DMSO-d6)δ7.90(s,1H,J＝7.9Hz,-NH-),5.32(qd,4H,J＝10.8, 5.3Hz,2×-CH＝CH-),4.26(dt,1H,J＝8.0,4.8Hz,-CHNH-),3.64(m,2H,-CH 2OH),2.74(t,2H, J＝6.4Hz,-CH＝CHCH 2CH＝CH-),2.13(t,2H,J＝7.4Hz,-CH 2CONH-),2.02(q,4H,J＝6.8Hz, 2×-CH2CH＝CH-),1.48(t,2H,J＝7.2Hz,-CH 2CH2CONH-),1.27(m,14H,7×-(CH2)-),0.86(t, 3H,J＝6.7Hz,-CH3)；13C NMR(101MHz,DMSO-d6)δ172.21,172.13,129.67,127.69, 61.43,54.49,35.00,30.88,29.04,28.73,28.71,28.63,28.60,26.63,26.58,25.20,25.18, 21.96,13.88.
(3) Oleanoyl glycine preparation (Compound 26)
Figure BDA0002132164030000062
Dissolving the obtained intermediate A1 in 45mL 65% ethanol, adding 3mL (21.52mmol) triethylamine, adding 0.643g (8.57mmol) glycine, and stirring at 25 ℃ in the dark for 72 h; after the reaction, the mixture was evaporated under reduced pressure to remove a large amount of ethanol, an appropriate amount of distilled water was added to the concentrate, pH was adjusted to 2 with concentrated HCl, an appropriate amount of ethyl acetate was added thereto, and the upper ethyl acetate layer was taken out, dried over anhydrous sodium sulfate, and concentrated. Silica gel column chromatography eluting with methylene chloride/methanol (30/1) gave 1.23g of product as a white powder in 52.14% yield.
1H NMR(400MHz,DMSO-d6)δ12.4(s,1H,-COOH-),8.02(d,1H,J＝5.5Hz,-NH-),5.29 (m,4H,2×-CH＝CH-),3.68(d,2H,J＝5.5Hz,-CH 2COOH),2.69(t,2H,J＝6.4Hz, -CH＝CHCH 2CH＝CH-),2.13(t,2H,J＝7.4Hz,-CH 2CONH-),2.02(q,4H,J＝6.8Hz, 2×-CH2CH＝CH-),1.48(m,2H,-CH 2CH2CONH-),1.17(m,14H,7×-(CH2)-),0.86(t,3H, J＝6.7Hz,-CH3)；13C NMR(101MHz,DMSO-d6)δ171.21,170.13,130.67,128.69,41.43, 44.49,28.73,28.71,28.63,28.60,26.63,26.58,21.96,13.88.
(4) Preparation of linoleoyl threonine (Compound 27)
Figure BDA0002132164030000071
Dissolving the obtained intermediate A1 in 45mL 65% ethanol, adding 3mL (21.52mmol) triethylamine, adding 1.00g (8.40mmol) threonine, and stirring at 25 deg.C in the dark for 72 h; after the reaction, the mixture was evaporated under reduced pressure to remove a large amount of ethanol, an appropriate amount of distilled water was added to the concentrate, pH was adjusted to 2 with concentrated HCl, an appropriate amount of ethyl acetate was added thereto, and the upper ethyl acetate layer was taken out, dried over anhydrous sodium sulfate, and concentrated. Silica gel column chromatography eluting with methylene chloride/methanol (10/1) gave 1.41g of product as a yellow oil in 52.87% yield.
1H NMR(400MHz,DMSO-d6)δ11.7(s,1H,-COOH-),7.32(d,1H,J＝8.8Hz,-NH-),5.26 (m,4H,2×-CH＝CH-),4.24(m,1H,-CH 2NH),4.74(m,1H,-CHOH-),2.63(t,2H,J＝6.4Hz, -CH ＝CHCH2CH＝CH-),2.12(t,2H,J＝7.8Hz,-CH2CONH-),2.08(m,4H,2×-CH 2CH＝CH-), 1.57(m,14H,7×-(CH2)-),0.96(d,3H,J＝6.7Hz,-CH3)；13C NMR(101MHz,DMSO-d6)δ 172.21,172.13,129.67,127.69,66.43,57.49,35.00,31.88,25.04,22.73,22.71,21.63, 20.60,13.88.
(5) Preparation of linoleoyl alanine (Compound 12)
Figure BDA0002132164030000072
Dissolving the obtained intermediate A1 in 45mL 65% ethanol, adding 3mL (21.52mmol) triethylamine, adding 0.75g (8.42mmol) alanine, and stirring at 25 deg.C in the dark for 72 h; after the reaction, the mixture was evaporated under reduced pressure to remove a large amount of ethanol, an appropriate amount of distilled water was added to the concentrate, pH was adjusted to 2 with concentrated HCl, an appropriate amount of ethyl acetate was added thereto, and the upper ethyl acetate layer was taken out, dried over anhydrous sodium sulfate, and concentrated. Silica gel column chromatography eluting with dichloromethane/ethyl acetate (20/1) gave 1.14g of product as a pale yellow oil in 46.40% yield.
1H NMR(400MHz,DMSO-d6)δ12.37(s,1H,-COOH),8.04(d,J＝7.4Hz,1H,，-NH-), 5.32(dt,J＝9.9,6.6Hz,4H，2×-CH＝CH-),4.18(t,J＝7.3Hz,1H,-CHNH-),2.74(t,J＝6.3 Hz,2H,，-CH＝CHCH2CH＝CH-),2.04(dt,J＝28.5,7.2Hz,6H,，-CH2CONH-),1.47(t,J＝ 7.1Hz,2H,，-CH2CH2CONH-),1.25(t,J＝7.5Hz,17H,7×-(CH2)-),0.86(t,J＝6.7Hz,3H, -CH3)。13C NMR(101MHz,DMSO-d6)δ174.25，171.88,129.67,127.69,47.26,34.95, 30.87,29.02,28.71,28.68,28.58,28.55,26.62,26.58,25.18,25.15,21.95,17.17, 13.87。
(6) Linoleoyl cysteine (Compound 11)
Figure BDA0002132164030000081
Dissolving the obtained intermediate A1 in 45mL 65% ethanol, adding 3mL (21.52mmol) triethylamine, adding 1.00g (8.25mmol) cysteine, and stirring at 25 deg.C in the dark for 72 h; after the reaction, the mixture was evaporated under reduced pressure to remove a large amount of ethanol, an appropriate amount of distilled water was added to the concentrate, pH was adjusted to 2 with concentrated HCl, an appropriate amount of ethyl acetate was added thereto, and the upper ethyl acetate layer was taken out, dried over anhydrous sodium sulfate, and concentrated. Silica gel column chromatography with dichloromethane/methanol (20/1) as mobile phase gave 1.26g of product as a pale yellow oil with a yield of 46.75%.
1H NMR(400MHz,DMSO-d6)δ12.40(s,1H,-COOH),8.04(d,J＝7.4Hz,1H,-NH-), 5.33(m,4H,，2×-CH＝CH-),4.18(t,J＝7.3Hz,1H，-CHNH-),2.74(t,J＝6.3Hz,2H, -CH＝CHCH2CH＝CH-),2.04(dt,J＝28.5,7.2Hz,6H,，2×-CH2CH＝CH-),1.47(t,J＝7.1Hz, 2H,-CH2CH2CONH-)，),1.26(q,J＝8.1,7.1Hz,14H,7×-(CH2)-),0.86(t,J＝6.8Hz,3H, -CH3)。
(7) Linoleoyl tyrosine (Compound 14, named NITyr)
Figure BDA0002132164030000082
Dissolving the obtained intermediate A1 in 45mL 65% ethanol, adding 3mL (21.52mmol) triethylamine, adding 1.50g (8.27mmol) tyrosine, and stirring at 25 ℃ in the dark for 72 h; after the reaction, the mixture was evaporated under reduced pressure to remove a large amount of ethanol, an appropriate amount of distilled water was added to the concentrate, pH was adjusted to 2 with concentrated HCl, an appropriate amount of ethyl acetate was added thereto, and the upper ethyl acetate layer was taken out, dried over anhydrous sodium sulfate, and concentrated. Silica gel column chromatography with dichloromethane/methanol (100/1) as mobile phase gave 0.98g of product as a pale yellow oil in 31.60% yield.
1H NMR (400MHz, DMSO-d6) δ 12.53(s,1H, -COOH),9.19(m,1H, -OH),8.02(d, J ═ 8.1Hz,1H, -NH-),7.00(m,2H, benzene ring 2 ', 6' -H),6.64(m,2H, benzene ring 3 ', 5' -H),5.32(m,4H, 2 × -CH ═ CH-),4.33(ddd, J ═ 9.5,8.0,4.8Hz,1H, -CHNH-),2.74(t, J ═ 6.3Hz,2H, Ph-CH ═ CH —),2.74(t, J ═ 6.3Hz,2H, Ph-CH ═ CH —)2-), 2.02(p,J＝6.8Hz,6H，-CH2CONH-,2×-CH2CH＝CH-),1.40(t,J＝7.3Hz,2H， -CH2CH2CONH-),1.27(m,14H，7×-(CH2)-),0.85(t,J＝6.8Hz,3H，-CH3)。
The beneficial effects of the compounds of the present invention are demonstrated by the following experimental examples.
Experimental example 1 linoleoyl tyrosine (NITyr) of the present invention antagonizes A β -induced cytotoxicity of PC12
1. Experimental methods
1.1 cell culture and grouping
PC12 cells were cultured in DMEM high-sugar medium containing 10% fetal bovine serum at 37 ℃ in a CO2 incubator with a volume fraction of 5%, and passaged by digestion with 0.25% trypsin. The experimental groups were as follows: normal control group (no Α β damage); ② A beta damage group (adding A beta); ③ NITyr groups (both Abeta and NITyr) with different concentrations (1 mug/mL, 5 mug/L, 10 mug/L, 20 mug/L); fourthly, 3MA + NITyr group (simultaneously adding Abeta, NITyr and autophagy inhibitor 3 MA); (V) autophagy inhibitor 3MA group (simultaneously adding Abeta and 3 MA).
1.2MTT assay
And (3) inoculating PC12 cells in logarithmic growth phase into a 96-well plate, arranging 8 multiple wells in each well, treating according to different experimental requirements, adding 10 mu L of MTT into each well after treatment, incubating at 37 ℃ for 4h, sucking out liquid in each well after completion, and discarding. Then, 100. mu.L of DMSO was added to each well, the mixture was shaken for 10min, and the absorbance (OD) of each well was measured by a microplate reader (450 nm). The average number of 8 wells was taken and the cell viability was calculated according to the formula. The formula is as follows: cell viability/%, treatment OD/control OD × 100%, was repeated 3 times.
1.3 establishment of PC12 cell A beta model
The results in fig. 1 show that after 100 μ g/mL of a β treated cells for 12, 24, 48 and 72h, the a β group had significantly decreased cell viability compared to the normal group, with significant differences (P <0.05, P <0.01, P <0.001), and cell viability gradually decreased with the time of a β treatment. 5. After 10, 20, 50, 100 and 200 mug/mL of A beta treatment cells are treated for 24 hours, the cell viability is gradually reduced along with the increase of the concentration, and the significant differences are all found under 50-200 mug/mL of A beta treatment (P <0.05, P <0.01 and P < 0.001).
The model was successfully established and subsequent experiments selected 100. mu.g/mL of A.beta.to treat PC12 cells for 24 h.
2. Results of the experiment
2.1 inhibition of A β -induced cytotoxicity of NITyr
PC12 cells were treated with 100. mu.g/mL of A.beta.for 24h, while the cells were treated with different concentrations of NITyr. The results in FIG. 2 show that cell viability increases gradually with increasing concentration of NITyr, with 5 and 10 μmol/L of NITyr providing a significant increase in cell viability, statistically significant (P <0.05, P <0.01) compared to the A β group.
The subsequent experiment selects 5 mu mol/L NITyr for development.
2.2 the Effect of the autophagy inhibitor 3MA on reversing the cellular viability of NITyr
Cells were treated with 5. mu. mol/L of drug. The results in fig. 3 show that 3MA alone has no change in cell viability compared to the normal group. Compared with NITyr, the 3MA + NITyr group has obviously reduced cell activity and has significant difference (P < 0.05); the combination of the autophagy inhibitor 3MA and the NITyr is proved to have obvious reversal effect on the cell activity increased by the NITyr.
Experimental example 2 study on inhibition of APP/PS1 transgenic mice by NITry on behavioral abnormalities
1. Experimental methods
1.1 open field experiment
The mouse is placed in the middle of the open box to freely move for 5min, and the automatic camera records the process of mouse tracking, the total movement distance in the box and the movement time of the mouse in corners, edges and central areas of the box.
1.2 fatigue rod rotating instrument experiment
The fatigue rod rotating instrument is used for carrying out rod rotating training on the mouse for 3 times every day, so that the mouse can basically move correspondingly along with the rod rotating within 5min, and the phenomenon that the mouse falls off due to the loss of balance on the rod rotating is avoided. The mice were forced to move on the rods at a speed of 30 r/min. The residence time on the rotarod was recorded for each group of mice.
1.3 Water maze experiment (Morris water maze, MWM)
The water maze is divided into 4 quadrants, and an organic glass platform (10cm multiplied by 30cm) is placed in one quadrant and hidden under the water surface by 1-2 cm. The mouse is placed into water from any quadrant, the automatic camera tracks the mouse track when the mouse enters the water, and the mouse track process and the time for searching the underwater platform are recorded. The water maze experiment mainly comprises a positioning navigation experiment and a space exploration experiment. Firstly, a positioning navigation experiment mainly trains a mouse and tests the learning and memory ability of the mouse. 1 day before the experiment, each mouse was freely placed in water for 60s to acclimatize. In the 1 st to 14 th days of the experiment, the mice are respectively put into the water from 4 water inlet points to the pool wall for a plurality of times, and the time for finding the platform hidden under the water is recorded by utilizing a water maze analysis system. If the mouse does not find a platform within 60s, it is directed to stay on the platform for 20 s. The spatial learning and memory ability of the mice was evaluated by calculating the average time each group of mice reached the platform daily. And secondly, a space exploration experiment detects the memory capacity of the mouse on the spatial position of the platform after training. The experiment is based on a water maze experiment device for removing a platform after a positioning navigation experiment, a mouse is placed into a water pool at 1 optional water entry point on the 15 th day of the positioning navigation experiment, the swimming track of the mouse in 60s is recorded, the time of the mouse in each quadrant is automatically analyzed by software, and the capability of the mouse to remember the platform hidden under the water surface is inspected. 1.4 packet setup
Normal group: normal mice, not treated with NITyr;
APP/PS1 group: APP/PS1 group mice, not NITyr treated;
NITyr group: APP/PS1 group mice were treated with 5,20 and 50mg/kg NITyr gavage for 2 months, respectively.
2. Results of the experiment
2.1 open field experiment
The total course of APP/PS1 group mice in the open field experiment is obviously shortened compared with normal group mice, and the statistical significance is achieved (P is less than 0.05, and figure 4); compared with the APP/PS1 group, the NITyr group has no significant difference in the total movement path of the mice. Meanwhile, the mouse has no statistical significance in the open field center, edge, corner and other areas.
2.2 fatigue rod rotation experiment
As shown in fig. 5, the mice stayed on the rotarod for a significantly longer period of time as the training period was extended. By the 4 th day of training, the time of staying on the rotating rod of the APP/PS1 group mice is obviously reduced compared with the normal group mice, and the statistical significance is achieved (P is less than 0.05); when training is carried out to the 5 th day, compared with the APP/PS1 group, the 20 and 50mg/kg NITyr groups can obviously improve the time of the transgenic mice staying on the rotating rod, and have statistical significance (P is less than 0.05); at day 7 of training, the 20 and 50mg/kg NITyr groups significantly increased the time that transgenic mice stayed on the rotarod compared to the APP/PS1 group, which was statistically significant (P < 0.01).
2.3 MWM
With the increase of the training days, the searching time of the mouse is continuously shortened by learning and memorizing the position of the underwater platform. When the mouse is trained to the 4 th day, compared with the APP/PS1 group of mice, the time for searching the underwater platform in the normal group is gradually shortened, and the mouse has statistical significance (P is less than 0.05, and figure 6A), and when the mouse is trained to the 5 th day, compared with the APP/PS1 group of mice, the time for searching the underwater platform in the 50mg/kg NITyr group is further shortened, and the mouse has statistical significance (P is less than 0.05, and figure 6A); at day 6 of training, the 20 and 50mg/kg NITyr groups significantly shortened the time for the transgenic mice to find the underwater platform compared to the APP/PS1 group, which was statistically significant (P <0.05, P <0.01, FIG. 6A).
After training to day 7, the platform was removed for exploratory experiments. As shown in FIG. 6B, the residence time in quadrant III was significantly increased for the NITyr group (20mg/kg, 50mg/kg), which was statistically significant (P <0.05, P < 0.01).
EXAMPLE 3 Effect of linoleoylserine (NIS, Compound 15 of the present invention) on A.beta.induced PC12 cell injury
1. Experimental methods
1.1 cell culture and grouping
PC12 cells were cultured in DMEM high-sugar medium containing 10% fetal bovine serum at 37 ℃ in a CO2 incubator with a volume fraction of 5%, and passaged by digestion with 0.25% trypsin. The experimental groups were as follows: normal control group (no Α β damage); ② A beta damage group (adding 100 mug/mL A beta); ③ NIS groups (plus both Abeta and NIS) at different concentrations (1. mu. mol/L, 5. mu. mol/L).
1.2 immunofluorescence
Clean and dry coverslips were placed in six well plates with cell density adjusted to 20 x 104after/mL, CO2Culturing for 24h in an incubator; the prepared medicine is added, 1 mL/hole, and the cells are continuously cultured for 24 h. After the culture medium in the six-hole plate is sucked out by a pipette gun in an adherent way, adding PBS to soak and wash the cells for 3 times for 3 min; add 1mL of 4% paraformaldehyde per wellSucking out after 15min, adding PBS to soak and wash the cells for 3 times for 3 min; adding 1mL of 0.5% Triton X-100 (prepared by 1X PBS) prepared in advance into each hole, and standing at room temperature for 20min to allow cell membrane to permeate; sucking out the permeation solution, adding 1mL of PBS into each hole, sucking residual Triton X-100, and repeatedly washing for 3 times for 3 min; sucking residual PBS in the plate by using clean filter paper, dripping 1mL of 5% BSA (prepared by 1X TBST) into each hole, shaking the culture plate lightly, and standing and sealing for 30min at room temperature; recovering the confining liquid, sucking the residual confining liquid through clean filter paper, slightly dropping 600 mu L of diluted DAPI (1 mu g/mL, prepared by RO water) into each hole, standing and incubating overnight in a refrigerator at 4 ℃, and washing away the residual DAPI staining liquid for 5min by 4 times through TBST; after observing the cell image by adopting a BX63 upright fluorescence microscope, the images are collected under a 10-time objective lens.
2. Results of the experiment
As shown in FIG. 7, it was found by immunofluorescence assay that the blue light was weak in the A.beta.group compared to the control group, indicating that the addition of A.beta.decreased the number of cells and the nucleus shriveled. However, both NIS groups at 1. mu. mol/L and 5. mu. mol/L reversed the above phenomenon, and as NIS concentration increased, fluorescence increased and cell viability increased. The compound 15 can reverse the damage of the PC12 cells induced by the A beta, so that the cell activity is obviously improved.
Experimental example 4 NIS inhibition of brain tissue A beta level of APP/PS1 transgenic mice
1. Experimental methods
1.1 grouping. Control group: normal mice, no NIS treatment; APP/PS1 group: APP/PS1 group mice, without NIS treatment; ③ NSS group: treating with NIS intragastric administration for 2 months.
1.2ELISA assay for A.beta.42And Abeta40The content of (A): perfusing with normal saline to remove blood to obtain mouse brain tissue sample, stripping hippocampal tissue, adding intermediate RIPA protein lysate, homogenizing at 4 deg.C in ice bath, centrifuging at 12000 r/min for 15min, collecting supernatant as sample to be tested, and using in Abeta42And Abeta40The detection and the operation steps refer to the Invitrogen kit instruction.
2. Results of the experiment
As shown in FIG. 8, withcomparison of control groups, A β in hippocampal tissue of brain in APP/PS1 group42And Abeta40The content of (A) is obviously increased, and the difference has statistical significance (P is less than 0.01 and P is less than 0.001). Compared with APP/PS1 group, NIS group can significantly reduce A beta42And Abeta40The difference has statistical significance (P is less than 0.05, and P is less than 0.01). The compound 15 of the invention is shown to be capable of significantly reducing the A beta level of APP/PS1 transgenic mice with AD.
In conclusion, the invention provides a compound shown in formula I with a novel structure, which can obviously inhibit A beta-induced cytotoxicity, can obviously reduce the A beta level of an APP/PS1 transgenic mouse with AD, improves the learning and memory capacity of the APP/PS1 transgenic mouse with AD, and has good application prospects in preparation of a beta-amyloid inhibitor and a medicament for preventing or treating Alzheimer's disease.

Claims (3)

1. Use of a compound or salt thereof for the preparation of an amyloid beta inhibitor:
Figure DEST_PATH_IMAGE001
Figure 304226DEST_PATH_IMAGE002
。
2. use according to claim 1, characterized in that: the amyloid beta inhibitor is a medicament for preventing or treating neurodegenerative diseases.
3. Use according to claim 2, characterized in that: the neurodegenerative disease is Alzheimer's disease.
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