WO2021143812A1 - METHOD FOR PREPARING N-(β-L-RHAMNOPYRANOSYL)FERULAMIDE AND USE THEREOF - Google Patents

Abstract

The present invention relates to a method for preparing compound I, N-(β-L-rhamnopyranosyl)ferulamide, and the use of compound I in the preparation of a medicament for alleviating or treating mitochondrial dysfunction and Aβ-induced mitochondrial dysfunction in a subject, treating depression, and improving or enhancing cognitive function.

Description

A kind of preparation method and application of N-(β-L-rhamnanopyranosyl) ferulic acid amide

Priority claim

This application claims the priority of Chinese patent application 202010050922.9 filed on January 17, 2020, which is incorporated herein by reference in its entirety.

Technical field

The present invention relates to a preparation method of a medicine, in particular, the present invention relates to a preparation method and application of N-(β-L-rhamnanopyranosyl) ferulic acid amide.

Background technique

Mitochondria are dynamic organelles in eukaryotic cells, which play an important role in ATP production, cellular calcium buffering and apoptosis. Mitochondrial DNA gene mutations can cause damage to the mitochondrial reactive oxygen species (ROS) scavenging function, cause ROS to accumulate in the mitochondria, cause mitochondrial oxidative damage, and may cause a series of changes in tissues and organs. SIRT3, a NAD-dependent histone deacetylase that mainly exists in mitochondria, can deacetylate the subunit proteins of the mitochondrial respiratory chain complex by exerting its deacetylase activity, and promote mitochondria to provide energy for cells Function. SIRT3 is involved in mitochondrial energy metabolism and cell aging, and is a molecular target for the treatment of aging and age-related diseases.

In recent years, some studies have shown that amyloid-β (Aβ) enters the mitochondria through the translocase of the outer membrane complex, that is, mitochondria may also serve as the target of Aβ, leading to a decline in cognitive ability and memory.

At present, N-(β-L-rhamnopyranosyl) ferulic acid amide) may have antioxidant effect and treat or alleviate mitochondrial dysfunction, Aβ-induced mitochondrial dysfunction, and improve cognitive ability have not been reported yet. Patent CN110117302A discloses the preparation method of N-(β-L-rhamnanopyranosyl) ferulic acid amide, which uses 1-amino-2,3,4-O-triacetyl rhamnose and (4 -O-TBS)-Ferulic acid chloride is used as raw material to obtain N-(β-L-rhamnanopyranosyl) ferulic acid amide. In the reaction, 1-amino-2,3,4-O-tri Acetyl rhamnose is a raw material, has high cost, many synthesis steps, difficult product purification, unsuitable for large-scale production, and relatively low reaction yield.

The synthetic route is as follows:

In view of the above-mentioned problems, the present invention provides a preparation method and application of N-(β-L-rhamnanopyranosyl) ferulic acid amide. The raw materials are cheap and easy to obtain, the reaction conditions are mild, the conversion rate is high, and the reaction steps are few. , High yield, high product purity, and applied to alleviate or treat mitochondrial dysfunction, alleviate or treat Aβ-induced mitochondrial dysfunction, improve or improve cognitive ability, etc.

Summary of the invention

The purpose of the present invention is to provide a method for preparing N-(β-L-rhamnanopyranosyl) ferulic acid amide and its medical use.

The present invention provides a method for preparing the compound N-(β-L-rhamnanopyranosyl) ferulic acid amide of formula I, which comprises the following steps:

1) Compound 2 is reacted with compound 3 in the presence of a base to obtain compound 1;

2) Compound 1 undergoes a deprotection reaction under the conditions of a deprotecting agent to obtain compound I;

The synthetic route is as follows:

The structural formula of compound 3 is as follows:

in,

P is selected from All, Boc, TMS, TES, TBS, TIPS, TBDPS, THP, MOM, MTM, MEM, BOM, SEM, EE, Bn, PMB, Cbz, DMB and Tr; X is selected from Cl and Br.

In a preferred embodiment, the reaction temperature in step 1) is -25°C-100°C, and the reaction solvent is selected from methanol, ethanol, propanol, isopropanol, tert-butanol, n-butanol, pyridine, and dichloromethane. Methane, tetrahydrofuran, 2-methyltetrahydrofuran (2-MeTHF), water, or any combination thereof.

In a preferred embodiment, the reaction temperature in step 2) is -5°C-60°C, and the reaction solvent is methanol, ethanol, propanol, isopropanol, tert-butanol, n-butanol, acetonitrile, 1,4 -Dioxane, tetrahydrofuran, dichloromethane or any combination thereof.

In a preferred embodiment, the base is selected from one or more of inorganic bases or organic bases, the molar ratio of compound 2 to base is 1:1-7; the molar ratio of compound 2 to compound 3 is 0.8-3:1-4; the molar ratio of compound 1 to the deprotection agent is 1:0.1-4.

In a preferred embodiment, the method further includes the step of subjecting the rhamnose compound to an ammonia source to perform a substitution reaction to obtain compound 2.

In a preferred embodiment, the reaction temperature of the substitution reaction is 15°C-100°C, the reaction time of the substitution reaction is 0.5-60h, and the reaction solvent is an alcohol solvent; the molar ratio of the rhamnose and the ammonia source The ratio is 1:1-10, preferably 1:1-7.

In a preferred embodiment, the method further comprises subjecting compound 5 to a hydroxyl protection reaction in an organic solvent to obtain compound 6, subjecting compound 6 to alkaline hydrolysis reaction to obtain compound 7, and subjecting compound 7 to halogenation reaction to obtain compound 3 steps,

The synthetic route is as follows:

In a preferred embodiment, the compound 5 undergoes a hydroxyl protection reaction with a hydroxyl protecting reagent under acid binding agent conditions to obtain compound 6, and the compound 6 undergoes alkaline hydrolysis under alkaline conditions to obtain compound 7, and compound 7 React with halogenated reagent to obtain compound 3.

The hydroxyl protection reaction is carried out in a suitable organic solvent, the reaction temperature is -5-70°C, and the reaction time of the hydroxyl protection reaction is 1-24 h; the molar ratio of the compound 5 to the acid binding agent is 1:1 -6, the molar ratio of the compound 5 to the hydroxyl protecting reagent is 1:1-5.

The alkaline hydrolysis reaction solvent is preferably an aqueous tetrahydrofuran solution, the alkaline hydrolysis reaction temperature is preferably room temperature, and the reaction time is 1-10 h; the molar ratio of the compound 5 to the base is 1:0.1-1.

The reaction temperature of the halogenation reaction is 10-60°C, the reaction time of the halogenation reaction is 1-10h, and the halogenation reaction solvent is selected from dichloromethane, acetonitrile or a combination thereof; the compound 7 and the halogenation reagent The molar ratio is 1:1-5.

The compound of formula 1 is a new compound, so the present invention also relates to the compound of formula 1 in another aspect:

In one aspect, this document provides N-(β-L-rhamnopyranosyl) ferulic acid amide and pharmaceutically acceptable salts thereof in preparation for alleviating or treating mitochondrial dysfunction in the cells of a subject Use in medicine.

In some embodiments, the mitochondrial dysfunction is Aβ protein, such as mitochondrial dysfunction induced by oligomers of Aβ42 polypeptide.

In some embodiments, the mitochondrial dysfunction includes, but is not limited to, increased levels of protein acetylation in mitochondria, increased levels of reactive oxygen species, decreased membrane potential, and/or decreased oxygen consumption; the drug is used to reduce mitochondria The level of protein acetylation, inhibits the reduction of membrane potential in mitochondria and/or inhibits the reduction of oxygen consumption in mitochondria.

In one aspect, provided herein is the use of N-(β-L-rhamnopyranosyl) ferulic acid amide and pharmaceutically acceptable salts thereof in the preparation of a medicament for increasing the activity or level of SIRT3 in a subject use. In some embodiments, the drug also enhances AMPK phosphorylation and/or enhances the activity or level of PGC-1. In some embodiments, the drug reduces the acetylation level of manganese superoxide dismutase (SOD2) and oligomycin sensitivity conferring protein (OSCP).

In one aspect, this article provides N-(β-L-rhamnanopyranosyl) ferulic acid amide and its pharmaceutically acceptable salt in preparation for reducing the acetylation and oxidation of mitochondrial protein in the cells of a subject. Use in medicine for stress levels or reactive oxygen levels. Preferably, the cells are nerve cells, such as SK-N-SH cells.

In one aspect, provided herein is the use of N-(β-L-rhamnanopyranosyl) ferulic acid amide and its pharmaceutically acceptable salt in the preparation of a medicament for the prevention, alleviation or treatment of depression. The drug can quickly and continuously exert a preventive, alleviating or therapeutic effect in a short-term or a long-term period, for example, within 1 hour of administration, and for a longer time, such as 8 hours, 16 hours, The effect can be maintained for 24 hours or more than 24 hours, for example, 2 days, 3 days, 7 days, 10 days, 15 days or even longer. Moreover, a single administration or preferably multiple repeated administrations can effectively maintain the prevention, alleviation or treatment of depression and its symptoms. In some embodiments, the drug exerts a preventive, alleviating or therapeutic effect within a short period of time after a single administration, and the short-term is no longer than 1 hour, no longer than 8 hours, no longer than 16 hours, no longer than 24 hours, or 24 hours to 72 hours. In some embodiments, the drug exerts a preventive, alleviating or therapeutic effect in a long-term period through multiple administrations, and the long-term period is no less than 3 days, no less than 4 days, no less than 5 days, no Less than 6 days, no less than 1 week, no less than 1 month, no less than 3 months, no less than 6 months, no less than 1 year, no less than 3 years, no less than 5 years or Longer. The dosing frequency can be once a day, once every 2 days, once a week, once every two weeks or a longer time interval. The dosing frequency can be determined by the physician according to the specific conditions of the patient or subject. The dosage is easily determined.

In one aspect, this article provides N-(β-L-rhamnanopyranosyl) ferulic acid amide and its pharmaceutically acceptable salts in preparation for preventing cognitive impairment in subjects, improving or enhancing The use of the subject's cognitive ability in drugs. In some embodiments, the decline in cognitive ability is response inhibition ability and/or memory ability.

In one aspect, provided herein is the use of N-(β-L-rhamnanopyranosyl) ferulic acid amide and pharmaceutically acceptable salts thereof in the preparation of a medicament for preventing, inhibiting or delaying senescence in a subject use. In some embodiments, the senescence is related to SIRT3.

In one aspect, provided herein is a method for preventing, alleviating or treating abnormal mitochondrial function in cells of a subject, which comprises administering to the subject N-(β-L-rhamnopyranosyl) ferulic acid amide Or a pharmaceutically acceptable salt thereof.

In one aspect, provided herein is a method for reducing mitochondrial protein acetylation, oxidative stress levels, or reactive oxygen species in cells of a subject, which comprises administering N-(β-L-pyran Rhamnosyl) ferulic acid amide or a pharmaceutically acceptable salt thereof.

In one aspect, provided herein is a method for preventing, improving or enhancing the cognitive ability of a subject, which comprises administering to the subject N-(β-L-Rhamnus pyran Glycosyl) ferulic acid amide or a pharmaceutically acceptable salt thereof.

In one aspect, provided herein is a method for inhibiting or delaying senescence in a subject, which comprises administering to the subject N-(β-L-rhamnopyranosyl) ferulic acid amide or its pharmaceutically Acceptable salt. In some embodiments, the senescence is related to SIRT3.

In one aspect, provided herein is N-(β-L-rhamnopyranosyl) ferulic acid amide or a pharmaceutically acceptable salt thereof to prevent cognitive impairment, improve or improve cognitive ability of subjects the use of.

Other aspects of the present invention are obvious to those skilled in the art due to the disclosure herein.

Description of the drawings

Figure 1 shows the compound I ¹ HNMR spectrum.

Figure 2 shows the HPLC profile of Compound I.

Figure 3 shows that treatment of SK-N-SH cells with PL171 for 24 hours has no effect on cell viability.

Figure 4 shows the effect of PL171 on the basic level of ROS.

Figures 5A-I show that PL171 promotes mitochondrial SIRT3 levels and their activity.

Figure 6A-L shows that PL171 promotes the expression of SIRT3 by enhancing AMPK/PGC-1.

Figures 7A-C show that PL171 inhibits Aβ42O-induced ROS production in SK-N-SH cells.

Figures 8A-E show that PL171 inhibits Aβ42O-induced reduction of MMP in SK-N-SH cells.

Figures 9A-D show that PL171 inhibits Aβ42O-induced decrease in oxygen consumption in SK-N-SH cells.

Figures 10A-B show the acetylation level of MnSOD in the mitochondrial lysate of SK-N-SH cells after SK-N-SH cells were pretreated with 30μM PL171 for 4 hours and then stimulated with 10μMAβ42O for 24 hours.

Figures 11A-C show that PL171 inhibits the reduction of SIRT3 and PGC-1α induced by Aβ42O.

Figures 12A-B show that PL171 improves Aβ42O-induced oxidative stress and mitochondrial dysfunction through SIRT3.

Figures 13A-B show that PL171 inhibits Aβ42O-induced cellular senescence through SIRT3 regulation.

Figures 14A-C show the antidepressant effects in mice evaluated by forced swimming immobility time (panel A, acute; panel B, medium and long period) and tail suspension experiment (panel C).

Figures 15A-B show a schematic diagram of the Stop-signal task model and its flow, and Figure 15C shows the administration schedule.

Figure 16A-B shows the effect of PL171 administration on Stop trial operation in the rat cognitive ability test model.

Figure 17A-B shows the effect of PL171 administration on Go trial operation in the rat cognitive ability test model.

Figure 18 shows the effect of intragastric administration of low, medium and high doses of PL171 on the time of forced swimming immobility 24h after a single dose of mice.

Detailed description of the invention

The present invention will be further described below in conjunction with specific implementation schemes and drawings. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

In this article, the addition, content and concentration of various substances are involved, and the percentages mentioned therein, unless otherwise specified, all refer to the percentages by mass.

In the embodiments herein, if no specific description is made for the reaction temperature or the operating temperature, the temperature usually refers to room temperature.

In the present invention, the terms "compound represented by structural formula n", "intermediate represented by structural formula n", and "compound n" have the same meaning, and all refer to the compound numbered n, where n refers to number I, 1, 2, 3, 4, 5, 6, 7. Similarly, compound I is sometimes referred to herein as PL171 or N-(β-L-rhamnopyranosyl) ferulic acid amide, and they have the same meaning.

The invention provides a method for preparing N-(β-L-rhamnopyranosyl) ferulic acid amide, which comprises the following steps:

1) Compound 2 is reacted with compound 3 in the presence of a base to obtain compound 1;

2) Compound 1 undergoes a deprotection reaction under the conditions of a deprotecting agent to obtain compound I;

The synthetic route is as follows:

The structural formula of compound 3 is as follows:

Wherein, P is selected from All (allyl), Boc (tert-butoxycarbonyl), TMS (trimethylsilyl), TES (triethylsilyl), TBS (tert-butyldimethylsilyl), TIPS (triisopropylsilyl), TBDPS (tert-butyldiphenylsilyl), THP (2-tetrahydropyranyl), MOM (methoxymethyl), MTM (methylthiomethyl) , MEM (methoxyethoxymethyl), BOM (benzyloxymethyl), SEM (trimethylsilylethoxymethyl), EE (ethoxyethyl), Bn (benzyl) , PMB (p-methoxybenzyl), Cbz (benzyloxycarbonyl), DMB (3,4-dimethoxybenzyl) and Tr (trityl); X is selected from Cl and Br; preferably, P is selected from TBS, Boc, Cbz and THP; X is selected from Cl; more preferably, P is selected from TBS.

In a preferred embodiment, the reaction temperature in step 1) is -25°C-100°C, and the reaction solvent is selected from methanol, ethanol, propanol, isopropanol, tert-butanol, n-butanol, pyridine, and dichloromethane. Methane, tetrahydrofuran, 2-methyltetrahydrofuran (2-MeTHF), water or a combination thereof; the reaction temperature of the step 2) is -5°C-60°C, and the reaction solvent is methanol, ethanol, propanol, and isopropanol , Tert-butanol, n-butanol, acetonitrile, 1,4-dioxane, tetrahydrofuran, dichloromethane or a combination thereof.

Preferably, the reaction temperature in the step 1) is -5°C-70°C, and the reaction solvent is methanol, dichloromethane, tetrahydrofuran or 2-methyltetrahydrofuran or a combination thereof; the reaction temperature in the step 2) is 0°C -30°C, the reaction solvent is methanol;

More preferably, the reaction temperature in step 1) is -5°C to 30°C; the reaction temperature in step 2) is 20°C to 30°C.

The progress of the reaction in the step 1) and the progress of the reaction in the step 2) can be monitored by conventional monitoring methods in the art (for example, TLC, HPLC, or NMR).

In a preferred embodiment, the reaction time in step 1) is 1-24 h; the reaction time in step 2) is 0.5-3 h; preferably, the reaction time in step 1) is 1-12 h; The reaction time of the step 2) is 0.5-2h.

In a preferred embodiment, the base is selected from one or more of inorganic bases or organic bases; the molar ratio of compound 2 to base is 1:1-7; the molar ratio of compound 2 to compound 3 is 0.8- 3:1-4; The compound 1 undergoes a deprotection reaction under the conditions of the deprotection agent to obtain compound I; the molar ratio of the compound 1 to the deprotection agent is 1:0.1-4.

Preferably, the molar ratio of the compound 2 to the base is 1:1-4; the molar ratio of the compound 2 to the compound 3 is 1:1-4; the molar ratio of the compound 1 to the deprotecting agent is 1:0.2-3.

More preferably, the molar ratio of the compound 2 to the base is 1:1.5-3; the molar ratio of the compound 2 to the compound 3 is 1:1-3; the molar ratio of the compound 1 to the deprotecting agent is 1:0.3-2.2 .

In the present invention, the inorganic base is selected from sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate, magnesium carbonate, lithium carbonate, lithium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, hydrogen One or more of strontium oxide, barium hydroxide, sodium hydroxide or potassium hydroxide; the organic base is selected from sodium methoxide, sodium ethoxide, N,N-diisopropylethylamine (DIEA), triethylamine , Diethylamine, tripropylamine, tri-n-butylamine, pyridine, N,N-lutidine, triethylenediamine, 1,5-diazabicyclo[5.4.0]undecene-5,1 One or more of ,5-diazabicyclo[4.3.0]nonene-5, 4-dimethylaminopyridine, N-methylmorpholine, and tetramethylethylenediamine.

Preferably, the base is selected from one of N,N-diisopropylethylamine (DIEA), pyridine, and sodium carbonate.

In a preferred embodiment, the deprotection agent is selected from the group consisting of tetrabutylammonium fluoride (TBAF), trifluoroacetic acid (TFA), palladium on carbon (Pd/C), palladium hydroxide on carbon (Pd(OH) ₂ / C), piperidine (Piperidine), hydrochloric acid methanol solution (HCl-MeOH), acetic acid (AcOH), formic acid (HCOOH), cesium fluoride (CsF), ammonium fluoride (NH ₄ F), potassium fluoride (KF) , Hydrofluoric acid-pyridine solution (HF·Py), hydrofluoric acid-triethylamine solution (3HF·TEA); preferably, the deprotecting agent is tetrabutylammonium fluoride ( TBAF), cesium fluoride (CsF), ammonium fluoride (NH ₄ F), potassium fluoride (KF), hydrofluoric acid-pyridine (HF·Py), hydrofluoric acid-triethylamine (3HF·TEA) One or more of.

The preparation method of the compound 1 includes the following post-processing steps: after the reaction, filtration, spin-drying, dissolution, and purification by column chromatography. The filtration, spin-drying, dissolution, and purification by column chromatography can be performed in accordance with the art This type of operation is carried out in the usual way.

The preparation method of the compound I preferably includes the following post-treatment steps: after the reaction is completed, spin-drying, dissolving, diluting, filtering, and drying, the spin-drying, dissolving, diluting, filtering, and drying can be performed in accordance with this type of operation in the art The conventional method is carried out.

In a preferred embodiment, the rhamnose compound undergoes a substitution reaction with an ammonia source to obtain compound 2.

In a preferred embodiment, the rhamnose compound and the ammonia source undergo the substitution reaction under acid binding agent conditions to obtain compound 2.

In a preferred embodiment, the reaction temperature of the substitution reaction is 15° C.-100° C., the reaction time of the substitution reaction is 0.5-60 h, and the reaction solvent is an alcohol solvent.

The alcohol solvent is selected from one or more of anhydrous methanol, anhydrous ethanol, isopropanol, and butanol.

Preferably, the reaction temperature of the substitution reaction is 20° C.-80° C., the reaction solvent is anhydrous methanol; the reaction time of the substitution reaction is 0.5-49 h.

In a preferred embodiment, the molar ratio of the rhamnose compound to the ammonia source is 1:1-10, and the molar ratio of the rhamnose compound to the acid binding agent is 1:1.5-4; Preferably, the molar ratio of the rhamnose compound to the ammonia source is 1:1-7, and the molar ratio of the rhamnose compound to the acid binding agent is 1:2-3.4.

In a preferred embodiment, the ammonia source is selected from one of ammonium bicarbonate, ammonium carbonate, ammonia water, and ammonia; preferably, the ammonia source is selected from one of ammonium bicarbonate, ammonium carbonate, and ammonia. kind.

The acid binding agent is an organic base or an inorganic base, and the organic base is diisopropylethylamine, diethylamine, tripropylamine, N,N-lutidine, triethylamine, tri-n-butylamine, Triethylene diamine, 1,5-diazabicyclo[5.4.0]undecene-5,1,5-diazabicyclo[4.3.0]nonene-5,4-dimethylaminopyridine , Pyridine, N-methylmorpholine, one or more of tetramethylethylenediamine; the inorganic base is sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, magnesium hydroxide, hydrogen One or more of calcium oxide, strontium hydroxide, barium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, lithium carbonate, magnesium carbonate, sodium bicarbonate, and potassium bicarbonate.

Preferably, the acid binding agent is triethylamine.

In a preferred embodiment, the rhamnose compound undergoes substitution reaction in an ammonia-alcohol solution to obtain compound 2;

The mass fraction of ammonia in the ammonia-alcohol solution is 8-20%; preferably, the mass fraction of ammonia in the ammonia-alcohol solution is 10-17%.

In a preferred embodiment, the rhamnose compound is selected from one or more of L-rhamnose and D-rhamnose; the L-rhamnose includes α-L-rhamnose, β-rhamnose, L-rhamnose; the D-rhamnose includes α-D-rhamnose and β-D-rhamnose; the α-L-rhamnose includes anhydrous α-L-rhamnose ( CAS: 6014-42-2), α-L-rhamnose monohydrate (CAS: 6155-35-7); the β-L-rhamnose includes anhydrous β-L-rhamnose (CAS: 6155-36-8), β-L-rhamnose monohydrate;

Preferably, the rhamnose compound is selected from L-rhamnose;

More preferably, the rhamnose compound is selected from α-L-rhamnose monohydrate (CAS:6155-35-7).

The more preferred synthetic route is as follows:

The preparation method of the compound 2 preferably includes the following post-treatment steps: after the reaction is completed, spin-drying and recrystallization; the spin-drying and recrystallization can be carried out according to conventional methods of this type of operation in the art.

The progress of the substitution reaction can be monitored by conventional monitoring methods in the art (for example, TLC, HPLC or NMR).

In a preferred embodiment, the compound 3 is obtained from compound 5 as a starting material, and is obtained through hydroxyl protection, alkaline hydrolysis, and halogenation. The synthetic route is as follows:

In a preferred embodiment, the compound 5 undergoes a hydroxyl protection reaction with a hydroxyl protecting reagent under acid binding agent conditions to obtain compound 6, and the compound 6 undergoes alkaline hydrolysis under alkaline conditions to obtain compound 7, and compound 7 React with halogenated reagent to obtain compound 3.

The acid binding agent in the hydroxyl protection reaction is selected from one of pyridine, 2-picoline, quinoline, imidazole, triethylamine, morpholine, N,N-diisopropylethylamine (DIEA) Or multiple.

Preferably, the acid binding agent in the hydroxyl protection reaction is selected from N,N-diisopropylethylamine.

In the present invention, the hydroxy protecting reagent is a hydroxy protecting reagent known in the art, preferably tert-butyldimethylchlorosilane (TBSCl).

The hydroxyl protection reaction is carried out in a suitable organic solvent, the reaction temperature is -5-70°C, and the reaction time of the hydroxyl protection reaction is 1-24 h; the molar ratio of the compound 5 to the acid binding agent is 1:1 -6, the molar ratio of the compound 5 to the hydroxyl protecting reagent is 1:1-5.

The organic solvent is preferably selected from dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMAC), N-methylpyrrolidone (NMP), hexamethylphosphoramide (HMPA) ), one or more of dichloromethane (DCM).

Preferably, the reaction time of the hydroxyl protection reaction is 3-12h.

Preferably, the molar ratio of the compound 5 to the acid binding agent is 1:2-5, and the molar ratio of the compound 5 to the hydroxyl protecting agent is 1:1.5-4.

Preferably, the reaction temperature in the hydroxyl protection reaction is 20-30° C., and the reaction solvent is DCM.

The preparation method of the compound 6 preferably includes the following post-processing steps: after the reaction, extraction, washing, drying, filtration, spin drying, beating, filtering, drying, the extraction, washing, drying, filtering, spin drying, and beating , Filtration, and drying can be carried out in accordance with the conventional methods of this type of operation in the field.

In a preferred embodiment, the alkali hydrolysis reaction solvent is selected from tetrahydrofuran, water or a combination thereof; the alkali includes one or more of potassium carbonate, sodium carbonate, potassium hydroxide, and sodium hydroxide.

Preferably, the alkaline hydrolysis reaction solvent is tetrahydrofuran aqueous solution; the base is potassium carbonate; preferably, the alkaline hydrolysis reaction temperature is room temperature, and the reaction time is 1-10 h.

The volume ratio of tetrahydrofuran to water in the tetrahydrofuran aqueous solution is 1-50:1; preferably, the volume ratio of tetrahydrofuran to water in the tetrahydrofuran aqueous solution is 5-20:1.

In the alkaline hydrolysis reaction, the molar ratio of the compound 6 to the base is 1:0.1-1; preferably, the molar ratio of the compound 6 to the base is 1:0.15-0.5.

The preparation method of the compound 7 preferably includes the following post-processing steps: after the reaction, extraction, washing, drying, filtering, spin drying, beating, filtering, drying, the extraction, washing, drying, filtering, spin drying, and beating , Filtration, and drying can be carried out in accordance with the conventional methods of this type of operation in the field.

The reaction temperature in the halogenation reaction is 10-100°C, preferably 10-60°C, the reaction time of the halogenation reaction is 1-10h, and the halogenation reaction solvent is selected from methylene chloride, acetonitrile or a combination thereof; the compound 7 The molar ratio with the halogenated reagent is 1:1-5.

Preferably, the reaction temperature of the halogenation reaction is 15-50° C., the reaction solvent in the halogenation reaction is dichloromethane; the molar ratio of the compound 7 to the halogenation reagent is 1:1-3 .

In a preferred embodiment, the halogenating agent is selected from the group consisting of trimethylchlorosilane, trimethylbromosilane, triethylchlorosilane, tert-butyldimethylchlorosilane, phenyldimethylchlorosilane, oxalyl chloride, Acetyl chloride, phosphorus oxychloride, phosphorus pentachloride, phosphorus pentabromide, thionyl chloride, sulfonyl chloride or a combination thereof; preferably, the halogenated reagent is oxalyl chloride.

The preparation method of the compound 3 preferably includes the following post-treatment steps: after the reaction is completed, spin-drying, and the spin-drying can be carried out according to conventional methods of this type of operation in the art.

Activity and function of PL171

The inventors found that PL171 (ie, N-(β-L-rhamnopyranosyl) ferulic acid amide) can increase the expression or activity of SIRT3. In some embodiments, PL171 promotes the expression of the SIRT3 gene, and/or promotes the activity of the SIRT3 protein. Furthermore, the inventors found that PL171 can also increase AMPK phosphorylation and PGC-1α expression. Since it is known that AMPK-mediated PGC-1α is one of the transcription factors of SIRT3 gene, it is likely that PL171 can promote the expression and activity of PGC-1α and SIRT3 by promoting AMPK activity.

SIRT3, as used herein, belongs to the 7 members of the "sirtuin family" in mammals. It is an NAD-dependent histone deacetylase that mainly exists in mitochondria. There are two forms of SIRT3: a long chain of 44kDa and a short chain of 28kDa, which mainly play a role in the cell through the short-chain SIRT3. SIRT3 is involved in mitochondrial energy metabolism and cell aging, and is a molecular target for the treatment of aging and age-related diseases.

PGC-1α, as used herein, has the full name of Peroxisome Proliferator Activated Receptor-γ (PPAR-γ) Coactivator-1α. This protein and other transcription factors are involved in the regulation of oxidative phosphorylation, lipid metabolism and mitochondrial biosynthesis. It is known that SIRT3 is a transcription target of PGC-1α.

In this application, it was confirmed that PL171 can significantly increase the expression and activity of SIRT3 or prevent its decrease, thereby attenuating the neuronal defects induced by Aβ42O. When the activity of SIRT3 is blocked, all these effects are absent, indicating that PL171 works by targeting SIRT3.

Moreover, through further studies as described in the examples, it is found that PL171 can significantly increase the mRNA and protein levels of PGC-1α, and prevent the decrease of Aβ-induced protein, thereby repairing the mitochondrial energy supply damage caused by Aβ. Therefore, PL171 may promote the expression of SIRT3 through PGC-1α.

In the present invention, PL171 can not only prevent Aβ42O-induced oxidative stress and mitochondrial damage, but also inhibit Aβ42O-mediated cell senescence.

The effect of PL171 on improving or enhancing cognitive function

As used herein, the term "cognitive function" in a broad sense refers to the process by which a person acquires, encodes, manipulates, extracts and uses sensory input information, including attention, memory, perception, and thinking. Disorders of cognitive function generally refer to clinical syndromes of varying degrees of cognitive impairment caused by various reasons (from physiological aging to disturbance of consciousness). There are many manifestations, such as learning or memory disorders, executive function disorders, dementia, aphasia, apraxia, agnosia, and other changes in mental and neurological activities.

In order to test the effect of PL171 in preventing cognitive dysfunction, improving or enhancing cognitive function, the inventors adopted the Stop-signal task model, which is a commonly used response inhibition behavior model and is widely used in clinical practice. Assessment of patient cognitive function and laboratory animal research. This model is designed on the theoretical basis of the "horse racing model", which can test the animal's reaction inhibition ability, and to a certain extent also reflects the animal's learning and memory ability, decision-making reaction ability and sports reaction ability. Response inhibition ability, working memory and attention regulation together constitute the main component of executive function, which is an important cognitive function. Inhibition of response refers to the suppression of the impulse of action that has been formed, which is a key component of executive control; specifically, the suppression of response is the suppression of no longer needed or inappropriate behaviors so that people can perform various flexible and active actions in the external environment. The purpose of the behavioral response.

In the present disclosure, after PL171 was administered to normal rats, it was found that the stop signal response time (SSRT) was significantly shortened (the control group did not change), and the correct rate of Stop trial operation was significantly improved. This result showed that the PL171 drug significantly improved the rat’s Response inhibition ability.

All the above-mentioned features mentioned in this document, or the features mentioned in the embodiments can be combined in any way. Each feature disclosed herein can be replaced with any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only limited examples of equivalent or similar features, and it is obvious that the present invention is not limited thereto.

The positive and progressive effects of the present invention are: the preparation method of the present invention has cheap and easily available raw materials, mild reaction conditions, high conversion rate, high yield, simple post-treatment, low production cost, and high chemical purity of the prepared product. The prepared PL171 (i.e. N-(β-L-rhamnanopyranosyl) ferulic acid amide) can play a role in treating mitochondrial dysfunction, improving the level and activity of SIRT3, treating depression, and improving cognitive ability. .

Example

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the content described can be applied to the method of the present invention. The specific implementation and materials described in the article are for demonstration purposes only.

Example 1

Preparation of compound 7

Compound 5 (8.50g, 43.80mmol) was dissolved in DMF (60mL), imidazole (11.74g, 175.00mmol) was added, TBSCl (13.20g, 87.60mmol) was added in batches at 0°C, and then placed at 21°C and stirred 12 hours; TLC showed that the reaction was complete, the reaction solution was poured into water (300 mL×5), extracted with ethyl acetate, the organic layers were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and spin-dried to obtain a yellow solid. Petroleum ether: ethyl acetate = 3:1 beating to obtain a white solid 13g (compound 6), dried, and then added to the reaction flask, adding THF (42.5mL) and H ₂ O (4.3mL) system stirring, adding K ₂ CO ₃ (0.25eq, calculated relative to the amount of compound 6), the system was stirred and reacted at 25±5℃ for 3.5 hours. After the reaction was over, water (10ml) was added, stirred for 10min, and the pH of the system was adjusted to 4-5 with a 3% HCl aqueous solution. , Separate the layers, collect the organic layer, wash with 20% NaCl aqueous solution (10ml), let stand, separate, collect the organic layer, _{dry MgSO 4} , filter, concentrate under reduced pressure to dryness, add n-heptane (17ml) and ethyl acetate Ester (2ml), stirred at 20±5°C for 1h, filtered and dried to obtain compound 7, whose structure was ^{confirmed by 1} H NMR;

¹ H NMR (600MHz, CD ₃ OD) δ: 7.43-7.46 (d, 1H), 7.02 (s, 1H), 6.90-6.92 (d, 1H), 6.67-6.69 (d, 1H), 6.17-6.20 ( d, 1H), 3.68 (s, 3H), 0.84 (s, 9H), 0.00 (s, 6H).

Preparation of compound 3

At room temperature, dissolve compound 7 (13.00g, 39.77mmol) in dichloromethane (150mL), add thionyl chloride (14.19g, 119.31mmol) dropwise, reflux and stir for 1 hour after dripping, and the reaction solution is directly spin-dried , Dichloromethane was added, and then spin-dried to obtain 13.50 g of yellow oil (compound 3).

Preparation of compound 2

Add compound 4 (5.00g, 27.5mmol), 20ml of anhydrous methanol and ammonium bicarbonate (4.82g, 60.92mmol) in a 100mL polytetrafluoroethylene tank, then add triethylamine (9.25g, 91.38mmol) The reaction was put into an oil bath and heated to 65°C and stirred for 40 minutes; then the reaction was placed at 21°C and stirred for 48 hours; TLC monitored that the raw materials were not reacted completely, and the reaction solution was directly spin-dried to obtain a light yellow foamy solid 10.20 g (Compound 2), directly used in the next reaction.

Preparation of compound 1

Compound 2 (5.80g, 26.30mmol) was dissolved in methanol (50mL), sodium carbonate (8.36g, 78.91mmol) was added, and the mixture was stirred at 0°C for 10 minutes. Then compound 3 (17.21g, 52.64) dissolved in tetrahydrofuran was slowly added. mmol), then placed at 21°C and stirred for 12 hours, filtered, the filtrate was directly spin-dried, 100 ml of dichloromethane was added to dissolve the crude product, 100-200 mesh silica gel was added to mix the sample, and column chromatography (dichloromethane: methanol = 1: 0-10: 1) Purification to obtain 3.90 g (compound 1) of pale yellow foam solid, yield: 32.7%.

¹ H NMR (600MHz, CD ₃ OD) δ: 7.38-7.40 (d, 1H), 7.01 (s, 1H), 6.90-6.92 (d, 1H), 6.67-6.68 (d, 1H), 6.47-6.50 ( d, 1H), 5.11 (s, 1H), 3.69 (m, 1H), 3.68 (s, 3H), 3.36 (m, 1H), 3.20 (m, 1H), 3.15 (m, 1H), 1.13-1.14 (d, 3H), 0.84 (s, 9H), 0.00 (s, 6H).

MS (ESI) m/z: [M+Na] ⁺ 476.2073; [MH] ⁺ 452.2070.

Preparation of compound I

Dissolve compound 1 (3.90g, 8.60mmol) in methanol (50mL), add 1N tetrabutylammonium fluoride solution in tetrahydrofuran (8.60mL) dropwise at 0°C, stir at 21°C for 1 hour, TLC (Dichloromethane: methanol = 7:1) shows that the reaction is complete, the reaction solution is spin-dried, add as little methanol as possible to dissolve, and then add dichloromethane to dilute until a solid precipitates, then add the same amount of dichloromethane, continue to stir After 10 minutes, it was filtered, and the filter cake was washed with dichloromethane to obtain 2.90 g of a white solid product (Compound I), yield: 65.1%.

¹ H NMR (600MHz, CD ₃ OD) δ: 7.42-7.45 (d, 1H), 7.07 (s, 1H), 6.95-6.97 (d, 1H), 6.70-6.71 (d, 1H), 6.48-6.51 ( d, 1H), 5.16 (s, 1H), 3.80 (s, 3H), 3.73 (m, 1H), 3.41 (m, 1H), 3.25 (m, 2H), 1.19-1.20 (d, 3H).

MS (ESI) m/z: [M+Na] ⁺ 362.1208; [MH] ⁺ 338.1210.

Example 2

Preparation of compound 7

DCM (32mL), compound 5 (8.00g, 1.00eq), DIEA (16.97g, 3.0eq) were added to the reaction flask, the system was stirred at 20-25°C, and TBSCl (12.4g, 2.00eq). The system was stirred and reacted for 4 hours. The system was added to ice water (0-10°C, 16mL); the system was allowed to stand for liquid separation; the organic phase was washed with 1M HCl aqueous solution (16mL), and the pH of the system was adjusted to 5-6; the system was stirred, Let stand for liquid separation; the organic phase was concentrated under vacuum and reduced pressure until no fraction was distilled out of the system, and the residue compound 6 (yellow oil, 20.16 g crude product) was collected;

Compound 6 (yellow oil, 20.16g crude product) was dissolved in THF (32mL) in H ₂ O (1.6 mL), stirred, and Na ₂ CO ₃ (0.88g, 0.20eq) was added to the system in batches, at 25-30 Stir at ℃ for 4 hours, add H ₂ O (12 mL) to the system, add 1M HCl aqueous solution to the system at 25±5 ℃ to adjust the pH of the system to 4-5, separate the liquids, and extract the aqueous phase with ethyl acetate (12 mL) , The combined organic phase was washed with saturated NaCl aqueous solution (12 mL); liquid separation, the organic phase was dried with anhydrous sodium sulfate, filtered, the organic phase was concentrated under reduced pressure until no fractions were evaporated, and n-heptane (20 mL) and acetic acid were added to the system Ethyl acetate (4 mL), stirred, filtered, the filter cake was rinsed with a mixed solution of n-heptane and ethyl acetate (0.5V, n-heptane: ethyl acetate = 10:1), the solid was collected and dried to obtain compound 7 (11.2g; purity: 99.99%, yield 87.76%) off-white solid, the structure was ^{confirmed by 1} H NMR;

¹ H NMR: (CD ₃ OD, 400MHz) δ: 0.16 (s, 6H), 1.00 (s, 9H), 3.84 (s, 3H), 6.35 (d, J=15.89 Hz, 1H), 6.84 (d, J = 8.19 Hz, 1H), 7.07 (dd, J = 8.19, 1.96 Hz, 1H), 7.18 (d, J = 1.96 Hz, 1H), 7.61 (d, J = 15.89 Hz, 1H).

Preparation of compound 3

Add DCM (40mL), compound 7 (10.00g, 1.00eq), DMF (0.015g, 0.005eq) to the reaction flask, stir, add dropwise oxalyl chloride (6.17g, 1.50eq) at 15-25℃, system Stir at 15-25°C for 3 hours. After the reaction is complete, the system is concentrated under reduced pressure until no solvent is evaporated to obtain compound 3 (10.8g) as a yellow oil. The structure is confirmed by 1H NMR:

¹ H NMR: (CDCl ₃ , 400MHz) δ: 0.20 (s, 6H), 1.01 (s, 9H), 3.87 (s, 3H), 6.51 (d, J = 15.41 Hz, 1H), 6.89 (d, J = 8.19 Hz, 1H), 7.00-7.16 (m, 2H), 7.78 (d, J = 15.41 Hz, 1H).

Preparation of compound 2

_{Add 100 mL of NH 3} -MeOH (ammonia mass fraction 10%) into the reaction kettle, add compound 4 (20.0g) in batches, heat the system to 35-40°C, stir for 24h, the reaction is over, the system is transferred out, the system is reduced Press and concentrate to dryness, add 50mL of absolute ethanol to the system, concentrate the system to dryness, add 50mL of absolute ethanol to the system, stir the system at 5-15°C for 2 hours, filter, and dry the filter cake to obtain compound 2 (11.34g, yield (Yield 57.04%) is an off-white solid, and the structure of the product is confirmed by 1H NMR:

¹ H NMR: (D ₂ O, 400MHz) δ: 1.16-1.36 (m, 3H), 3.25-3.44 (m, 2H), 3.57 (br d, J = 9.03 Hz, 1H), 3.84 (br s, 1H) ), 4.30 (br s, 1H).

Preparation of compound 1

At room temperature, add 2-MeTHF (120mL), compound 2 (10.00g, 1.00eq), pyridine (7.27g, 1.50eq) to the reaction flask, stir, and cool the system to 0-5°C at 0-5°C A solution of compound 3 (20.03g, 1.00eq) in 2-MeTHF (0.10L) was added dropwise, and after the dropping was completed, the system was stirred at 0-5°C for 2 hours, and the reaction was completed. MeOH (10mL) was added to the system at 10-15°C, stirred, saturated brine (40mL) was added to the system, the system was separated, the organic phase obtained was concentrated to dryness under reduced pressure, and the crude product obtained was used MTBE (80mL) and n-heptane (40mL) Stir at 65-80°C for 2 hours, cool the system to 20-30°C, filter, collect the filter cake, and dry (40-50°C) to obtain compound 1 (19.00g, yield: 68.34%, purity : 99.99%) off-white solid, the structure of the product was ^{confirmed by 1} H NMR:

¹ H NMR: (CD ₃ OD, 400MHz) δ: 0.17 (s, 6H), 1.00 (s, 9H), 1.30 (d, J = 5.65 Hz, 3H), 3.33-3.41 (m, 2H), 3.51 ( s, 1H), 3.78-3.92 (m, 4H), 5.26 (d, J = 1.00 Hz, 1H), 6.65 (d, J = 15.69 Hz, 1H), 6.85 (d, J = 8.16 Hz, 1H), 7.08 (dd, J = 8.16, 1.88 Hz, 1H), 7.19 (d, J = 1.88 Hz, 1H), 7.55 (d, J = 15.56 Hz, 1H).

Preparation of compound I

Add compound 1 (8.00g, 1.00eq) and THF (80mL) into the reaction flask, stir, add TBAF (1.84g, 0.40eq) dropwise to the system at 15-25℃, and the system will react with stirring at 20-25℃ The reaction is over in 1 hour. The system was concentrated under reduced pressure until no liquid was evaporated to obtain a crude product of PL171 (yellow solid, 8.00 g).

¹ H NMR: (CD ₃ OD, 400MHz) δ: 1.30 (d, J = 5.62 Hz, 3H), 3.32-3.43 (m, 2H), 3.46-3.55 (m, 1H), 3.77-3.95 (m, 4H) ), 5.26 (d, J = 0.61 Hz, 1H), 6.59 (d, J = 15.65 Hz, 1H), 6.80 (d, J = 8.07 Hz, 1H), 7.06 (dd, J = 8.19, 1.83 Hz, 1H ), 7.16(d,J=1.71Hz,1H), 7.53(d,J=15.65Hz,1H).

Example 3: Experimental materials and experimental methods

The reagents and raw materials used in the present invention are all commercially available.

Aβ42O (Aβ42 oligomer) preparation

The Aβ42 polypeptide was treated with hexafluoroisopropanol (HFIP) and resuspended in dimethyl sulfoxide (DMSO), then diluted to 100μM in DMEM/F12 phenol red-free medium, centrifuged, and then at 4℃ Incubate for 24 hours under low temperature; wherein, Aβ42 peptide was purchased from Genicbio (A-42-T-2).

Cell culture

SK-N-SH cells were purchased from ATCC. The cell line was placed in a modified medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 0.1 mg/mL streptomycin, and cultured in a constant temperature incubator.

Measurement of cell viability

SK-N-SH cells ^{were seeded in a 96-well plate at 1×10 4} cells/well. After being treated with the specified concentration of PL171 for 24 hours, the Cell Titer-Glo luminescence assay (Promega, G7573) was used to detect cell survival. Value measured by BioTek Synergy NEO (Bio-Tek, USA). As shown in Figure 3, the survival rate of SK-N-SH cells was not affected by treatment with 30uM PL171 for up to 24h.

Mitochondrial separation

Inoculate SK-N-SH cells into a 60mm culture dish, wash the cells (1.5×10 ⁶ cells/dish) with PBS once, decompose with trypsin-EDTA solution and centrifuge at 200g for 10 minutes, discard the supernatant, and then Resuspend the pellet in cold PBS, centrifuge at 600g for 5 minutes at 4°C, then resuspend it with 1mL mitochondrial separation solution containing 100μM PMSF, incubate on ice for 10 minutes, and then draw the cell resuspension through a 1ml insulin needle. The homogenate was homogenized for 10 times and centrifuged at 600g for 10 minutes at 4°C. After collecting the supernatant, the supernatant was centrifuged at 11000g for 10 minutes at 4°C to obtain mitochondria, and then the mitochondrial lysate was analyzed by Western blot.

Reactive oxygen species (ROS) analysis

Use 2,7-dichloro-fluorescein diacetate (DCFH-DA) (Beyotime, S0033) as a probe to detect intracellular ROS levels, that is, SK-N-SH cells are placed in 1×10 ⁴ cells/well Density seeded in a 96-well plate, and then treated the cells with the specified concentration of Aβ42O or PL171 for 24 hours. The cells were incubated with 10μM DCFH-DA in serum-free and phenol red medium for 30 minutes, and the medium was placed at 37°C. In a humidified incubator containing 5% CO ₂ /95% air (v/v), the cells were washed twice with PBS, and then observed under a laser measurement and a confocal microscope (Operetta, Perkin Eimer, USA). Alternatively, cells in a 96-well black plate were treated with 1% Triton X-100 at 37°C for 10 minutes, and fluorescence was measured using BioTek SynergyNEO (Bio-Tek, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Mitochondrial ROS detection

The cells were pre-incubated for 4 hours with or without PL171, and then treated with 10 uM Aβ42O for 24 hours. At the end of the treatment, the cells were co-stained with 2.5 μM MitoSOX Red mitochondrial superoxide indicator (Invitrogen, M36008) and 3 μg/mL nuclear staining dye Hoechst (Beyotime, C1022) at 37°C for 20 min. BioTek SynergyNEO was used to record fluorescence signals at 510/580nm (MitoSOX) and 350/461nm (Hoechst). The MitoSOX fluorescence signal was normalized by Hoechst signal intensity.

Measurement of mitochondrial membrane potential

SK-N-SH cells were seeded into 96-well plates (Costar, 3904) at a density of 10,000 cells/well. Cells were treated with the indicated concentrations of Aβ _42-1 O Aβ42O or 4 or 24h or pretreated with the indicated concentrations of PL171, and reprocessing Aβ42O 24h, using the JC-1 kit (Beyotime, C2006) detect mitochondrial membrane potential of cells ( MMP) level; that is, add the mixed JC-1 staining solution to the cells at 37°C for 30 minutes, wash twice with the diluted JC-1 staining buffer, and observe the cells under the Zeiss Observer Z1 microscope. BioTek SynergyNEO (Bio-Tek, USA) was used to detect the fluorescence intensity at 490/530nm (green) for monomers and 525/590nm aggregates (red), and the membrane potential was expressed as the ratio of red/green fluorescence intensity.

Determination of Cellular Oxygen Consumption Rate

An Agilent Seahorse XFe24 cell energy metabolism analyzer was used to measure the oxygen consumption rate of SK-N-SH (neuroblastoma cells). First, the neuroblastoma cells at a density of 3 × 10 ⁴ and seed in 24 well plates, cells were pretreated 30uM PL171 4h, the corresponding control group given a solvent and then applied 10μM Aβ42O incubated 24h. Before measuring the oxygen consumption rate of cells, cells need to be placed in _{an incubator at 37°C without CO 2} and treated with a non-buffered culture medium containing 25 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate without bicarbonate. 45min. After measuring the basic cell oxygen consumption, oligomycin, FCCP uncoupling agent, rotenone, and finally antimycin A were added in sequence. The measured data is analyzed by Seahorse XFe24 software.

SA-β-gal measurement (determination of β-galactosidase activity)

Use a commercial kit (Beyotime, C0602) to measure cell senescence by SA-β-gal staining, and place SK-N-SH cells (5×10 ⁴ cells/well) in a medium containing 5% FBS in a 24-well plate Cultured in medium, treated with Aβ42O in the absence or presence of PL171 for 72h, then stained with SA-β-gal, and photographed and counted the blue-stained cells with a Zeiss Observer Z1 microscope. In each experiment, at least 10 different fields (60-100 cells/field) are counted.

Reverse transcription and real-time PCR

After treating the cells with the specified concentration of PL171, the ^{total RNA of 2×10 5} cells/well was extracted with the TRI reagent provided by sigma (T9424), and then reversed with the PrimeScript RT master mix of TakaRa (RR036B) After the reverse transcription reaction was performed, SYBR Green Qpcr master mix (ExCell Bio) was selected for real-time fluorescent quantitative PCR operation, and HPRT was used as the internal control.

Western Blot

The cells (1×10 ⁵ cells/well) were treated with PL171 for 24h or pretreated with PL171 for 4h and then treated with Aβ42O for another 24h. For mitochondrial lysate preparation, cells are seeded and mitochondria are isolated as previously described. Use 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the total cell lysate or mitochondrial lysate of neuroblastoma cells. The electrophoresis condition is 400mA constant current. , 4℃, 2h, after transfer, use 5% fat-free milk blocking solution containing 0.1% Tween-20 to block for 1 hour at room temperature. After the blocking is completed, incubate the primary antibody. The primary antibodies used include:

SIRT3, provided by the brand Cell Signaling Technology, item number 5490S;

ATP5A, brand Abclonal, article number A5884;

SIRT1, brand Proteintech, article number 13161-1-AP;

OSCP, brand Santa Cruz Biotechnology, item number sc-365162;

ATP5Ok139, brand Abcam, article number ab214339;

SOD2, brand Santa Cruz Biotechnology, item number sc-133134;

SOD2k68, brand Abcam, item number ab137037;

PGC-1α, brand Proteintech, article number 66369-1-Ig;

AMPKα rabbit monoclonal, brand Beyotime, catalog number AF1627;

Phospho-AMPKα, brand Beyotime, article number AA393;

Actin, brand Sigma, item number #A2066;

After overnight at 4℃, add HRP-conjugated secondary antibody, then incubate with ECL substrate, take pictures and analyze with imaging system.

Statistical Analysis

Analyze the data through Prism 6.0 (GraphPad Software Inc, San Diego, CA), use unpaired t-test (two-tailed) comparison between the two sets of data, and use single-factor variance combined with Bonferroni post-test, p <0.05 means a significant difference.

Example 4: PL171 improves mitochondrial SIRT3 level and activity

Mitochondrial protein acetylation is closely related to mitochondrial function. The effect of PL171 on the acetylation status of mitochondrial protein was tested. In short, SK-N-SH cells were treated with different concentrations of PL171 for 24 h, then mitochondria were separated, lysates were prepared, and total acetylation of mitochondrial proteins was measured by Western blotting using anti-acetylation antibody (Ac-k). The results are shown in Figure 5A, which indicates that PL171 dose-dependently reduced the effect of total acetylation of mitochondrial proteins.

The cells were also treated with PL171 at 30 μM for 0.5-24 h to observe the changing process of the degree of mitochondrial protein deacetylation. The results are shown in Fig. 5B, which shows that the acetylation of mitochondrial proteins of the cells is the lowest after 30 μM 171 treatment for 24 hours.

Since SIRT3 plays an important role in the deacetylation of mitochondrial proteins, the expression of SIRT3 in mitochondria was determined. The results of immunoblotting showed that PL171 increased mitochondrial SIRT3 by about 36% (see Figures 5C and 5F).

In order to detect SIRT3 activity, specific antibodies were used to detect the acetylation level of SIRT3 substrates, including manganese superoxide dismutase (SOD2) and oligomycin sensitivity conferring protein (OSCP). Immunoblotting was used to detect the acetylation levels at positions 68 and 139, respectively. Acetylation. The results showed that PL171 reduced the acetylation of MnSOD and OSCP in a dose-dependent manner, and 30 μM PL171 reduced the acetylation of MnSOD (SODk68/MnSOD) and OSCP (ATP5O/OSCP) by approximately 20% and 32%, respectively (see Figure 5C, D). , E); Pretreatment with SIRT3 inhibitor (SIRT3inh., 3-TYP, 20μM, 4h) significantly blocked the effect of PL171 (see Figure 5G, H, I).

The above results indicate that PL171 can protect mitochondrial function by increasing the level or activity of SIRT3 in mitochondria to promote mitochondrial protein deacetylation.

Example 5: PL171 promotes the expression of SIRT3 by enhancing AMPK phosphorylation-mediated PGC-1

SK-N-SH cells were treated with different concentrations of PL171 for 24h, and then the cells were collected to prepare lysates. PL171 dose-dependently increased the expression of SIRT3 in total cell lysates by about 25% at 30 μM, but had almost no effect on SIRT1 levels (see Figure 6A, B, G, H), indicating the specific effect of PL171 on SIRT3.

Treatment with PL171 for 24h significantly promoted the mRNA level of SIRT3 but not SIRT1 (see Figure 6C, 6I).

The expression of SIRT3 gene is controlled by the transcription factor PGC-1α involved in mitochondrial biogenesis. Therefore, the stimulation of PGC-1mRNA and protein expression by PL171 was tested. The results show that treatment with PL171 for 24h can promote PGC-1α mRNA and protein levels, indicating that PL171 may Promote the expression of SIRT3 by enhancing PGC-1 (see Figure 6D-F).

In addition, it is known that the activation of AMPK can stimulate CREB-mediated up-regulation of PGC-1 expression, thereby transferring to regulate the expression of SIRT3. Therefore, the effect of PL171 on AMPK was tested, and the results showed that PL171 can promote AMPK phosphorylation, and pretreatment with AMPK activity inhibitor compound C (compound C) can reduce the effect of PL171 on AMPK and inhibit the expression of SIRT3 (see Figure 6J- L).

Example 6: PL171 inhibits Aβ42O-induced ROS production in SK-N-SH cells

First, after treating human nerve cells SK-N-SH with PL171 for 24 hours, the basal ROS production was reduced by about 15% (see Figure 4, 30uM PL171). Aβ42O can induce the production of ROS, which can cause oxidative stress in neurons. Human nerve cells SK-N-SH were treated with different concentrations of Aβ42O for 24h, and the cell ROS levels were measured by staining with DCFH-DA. The results are shown in Figure 7A, which shows that Aβ42O induces an increase in ROS, but by pretreatment with PL171, The production was decreased in a dose-dependent manner, and 30 μM PL171 almost completely inhibited the production of ROS induced by Aβ42O. The results are shown in Fig. 7B.

In order to specifically detect mitochondrial ROS, the mitochondrial superoxide indicator MitoSOX was used. The results are shown in Figure 7C. The data showed that Aβ42O (10uM, 24h) increased mitochondrial ROS stimulation by about 26%, and pre-incubation with PL171 (30uM, 4h) could significantly inhibit this increase in stimulation.

These results indicate that PL171 protects nerve cells from Aβ42O-induced oxidative damage.

Example 7: PL171 inhibits the decrease of mitochondrial membrane potential (MMP) in SK-N-SH cells induced by Aβ42O

Aβ42O can induce the loss of MMP. The JC-1 probe is used to evaluate the MMP in SK-N-SH cells. The red fluorescence and green fluorescence represent the high and low permeability of the mitochondrial membrane, respectively, and this ratio can represent the change of MMP.

Compared with the control group, treatment with Aβ42O significantly reduced the ratio of red/green fluorescence (Figure 8A), indicating that MMP depolarization induced by Aβ42O, and Aβ42-1 as a negative control had no significant effect (Figure 8A); Aβ42O had no significant effect on MMP The effect of Aβ42O (10μM) was time- and dose-dependent, and Aβ42O (10μM) reduced MMP by about 12%, 32% and 36% at 8h, 16h and 24h, respectively (Figure 8B). Pretreatment with PL171 for 4h significantly prevented the reduction of MMP in SK-N-SH cells induced by Aβ42O (Figure 8C); Aβ42O (10μM, 24h) induced a 34% reduction in MMP, which was attenuated by pre-incubation with 30μMPL171 for 4h. 10%. When the pre-incubation period of PL171 was extended to 24h, the protective effect of PL171 was more significant (Figure 8D). At the same time, PL171 did not change MMP in cells without Aβ42O, while rotenone as a positive control produced a 37% reduction ( Figure 8E).

Example 8: PL171 inhibits the reduction of oxygen consumption in SK-N-SH cells induced by Aβ42O

Aβ accumulates in mitochondria, leading to ATP depletion, decreased respiratory rate, and decreased respiratory enzyme activity. The oxygen consumption rate (OCR) was analyzed using a hippocampal instrument to detect the effect of PL171 on mitochondrial function.

Compared with the control group, Aβ42O (10μM, 24h) OCR was impaired, and the presence of PL171 (30μM, 4h pretreatment) inhibited Aβ42O-induced mitochondrial damage (Figure 9A); Aβ42O base decreased respiration by 21%, which was pre-cultured with 30μM PL171 4h returned to the control level (Figure 9B); Aβ42O reduced ATP production by about 25%, while pretreatment with PL171 (30μM) for 4h restored the ATP level to a level similar to that of the control (Figure 9C); compared with the control group, Aβ42O The mitochondrial maximum respiration was impaired by 22%, and after PL171 pretreatment, the mitochondrial maximum respiration impairment could be completely inhibited (Figure 9D); the above data showed that PL171 can inhibit the reduction of oxygen consumption induced by Aβ42O, including ATP production, basal respiration and maximum Breath and maintain healthy mitochondrial function.

Example 9: PL171 inhibits the increase of Aβ42O-induced acetylation level in SK-N-SH cells

Pretreated with 30μM PL171 for 4h, and then treated with 10μMAβ42O for 24h, observe the acetylation level of MnSOD in the mitochondrial lysate of SK-N-SH cells

Mitochondrial protein acetylation is closely related to mitochondrial function. Aβ42O (10μM) increased the acetylation level of MnSOD, which was significantly down-regulated by pre-incubating with 30μM PL171 for 4h (Figure 10A, 10B). The data indicated that PL171 can promote SIRT3 function. Promote mitochondrial protein deacetylation to inhibit Aβ42O-induced mitochondrial dysfunction.

Example 10: PL171 inhibits the reduction of SIRT3 and PGC-1α induced by Aβ42O

Compared with the control group, Aβ42O (10μM, 24h) reduced the expression of SIRT3 and PGC-1α. Pretreatment with PL171 for 4h attenuated the decrease in the expression of SIRT3 and PGC-1 induced by Aβ42O. Pre-incubation with 30μM PL171 completely blocked The reduction of SIRT3 and PGC-1α expression caused by Aβ42O (Figure 11A, 11B, 11C).

Example 11: PL171 improves Aβ42O-induced oxidative stress and mitochondrial dysfunction through SIRT3

Compared with the control group, Aβ42O (10μM, 24h) reduced MMP by 32%, which was successfully prevented by PL171 (30μM, pre-incubation for 4h). In cells pretreated with SIRT3 inhibitor (20μM, 4h), Aβ42O reduced MMP 28%, while PL171 did not change (Figure 12A). PL171 inhibited the Aβ42O-mediated increase in ROS levels, which was attenuated when 3-TYP was simultaneously administered with PL171 (Figure 12B). These data indicate that PL171 mediates the protective effect of Aβ42O-induced oxidative stress and mitochondrial dysfunction through SIRT3.

Example 12: PL171 inhibits Aβ42O-induced cellular senescence through SIRT3 regulation

Through the staining of SA-β-gal, it was observed that Aβ42O (10μM, 72h) increased the number of SA-β-gal positive cells more than twice (Figure 13A); by pretreatment with PL171 for 4h, compared with the control group, 30μM PL171 reduced the number of SA-β-gal positive cells promoted by Aβ42O to the control level. In cells with 20μM 3-TYP (SIRT3 inhibitor), Aβ42O (10μM, 72h) resulted in the number of SA-β-gal positive cells The increase in Aβ42O was similar to that of cells without 3-TYP (Figure 13B). The co-treatment of PL171 and 3-TYP did not change the effect of Aβ42O. The data indicated that PL171 protected neuronal cells from Aβ42O-induced mitochondrial-associated cells by promoting SIRT3 activity. senescence.

Example 13: Evaluation of the acute antidepressant effect of PL171 in mice

1. Reagents and medicines:

The medium is three distilled water;

Fluoxetine hydrochloride (FLX): Tokyo Chemical Industry Co., Ltd., product number: F0750.

PL171

2. Animals: Healthy C57BL/6J mice, male, weighing 18-22g, from Shanghai Slack Laboratory Animal Co., Ltd., arrived at the Animal Breeding Center of Shanghai Institute of Materia Medica, Chinese Academy of Sciences before the experiment (Animal production license: SCXK9[沪]2004 -0002, use permit: SYXK[沪]2003-0029), and adapt to animal facilities for more than 3 days, 6 animals per cage. (Animal production license: SCXK9[沪]2004-0002, use license: SYXK[沪]2003-0029).

3. Method: Divide healthy C57BL/6J mice into 5 groups randomly, 10 mice in each group, which are vehicle group (three distilled water); control group (fluoxetine hydrochloride 20.0mg/kg); PL171 high and medium , Low-dose group (50.0, 15.0, 5.0mg/kg), intragastric administration once. During the experiment, the animals were free to eat and drink. After a single administration for 1 hour, the mice were placed in a container with a diameter of about 18 cm, a water depth of 18 cm, and a water temperature of 25°C. The swimming time of the mice was 6 minutes. Movement time (that is, the mouse stops struggling in the water, or the animal is floating, with only small limbs moving to keep the head floating on the water). This time is also known as the forced swimming immobility time, which is an indicator known in the art for determining the degree of depression. The shorter the time, the higher the activity of the mouse and the better the antidepressant effect.

4. Data on forced swimming immobility time

Table 1

5 Conclusion

The low, medium and high doses of PL171 can significantly reduce the immobility time of forced swimming in mice, and the antidepressant effect is obvious.

Example 14: Evaluation of the antidepressant efficacy of PL171 in mice after a single administration 24h

1. Reagents and medicines:

Solvent: 0.9% saline

Fluoxetine hydrochloride (Fluoxetine): Tokyo Chemical Industry Co., Ltd., product number: F0750

PL171: Central Research Institute of Shanghai Pharmaceutical Co., Ltd.

2. Animals: Healthy C57BL/6J mice, male, weighing 18-22g, from Shanghai Lingchang Biotechnology Co., Ltd., arrived at the Animal Feeding Center of Shanghai Institute of Materia Medica, Chinese Academy of Sciences before the experiment (Animal production license: SCXK[沪]2018 -0003, use license: SYXK[沪]2020-0042), and adapt to animal facilities for more than 3 days, 5 animals per cage.

3. Method: Divide healthy C57BL/6J mice into 6 groups randomly, 15 mice in each group, which are the vehicle group (0.9% saline); the control group (fluoxetine hydrochloride 10.0mg/kg); the control group (the wormwood hydrochloride) Sketamine 10.0 mg/kg); PL171 high, medium and low dose groups (50.0, 15.0, 5.0 mg/kg). Forced swimming was measured 24 hours after intraperitoneal administration in the control group, and forced swimming was measured 24 hours after intragastric administration in the other groups. During the experiment, the animals were free to eat and drink. After a single dose of 24 hours, the mice were individually placed in a cylindrical glass cylinder with a height of 30 cm and a diameter of 20 cm. The water depth in the tank was 15 cm, so that the mice could not escape. The feet and tail of the glass cylinder do not touch the bottom of the cylinder, and the water temperature is 23℃-25℃. A video was taken 6 minutes after the mice entered the water. Since most mice are very active in the first two minutes, the immobility time after 4 minutes is calculated (criteria of immobility: the mouse stops struggling in the water, does not move, and is in order to maintain balance or display. Small limb movements in a floating state. Mice in each group operate in parallel). This time is the time of forced swimming immobility.

4. Data on forced swimming immobility time

All data analysis is done using spss 22 data processing software. One-way analysis of variance (One-way anova) is used, and the post-hoc LSD method is used for multiple comparison verification results. The data is represented by Mean±sem. When p<0.05, one asterisk is marked; when p<0.01, two asterisks are marked; when p<0.001, three asterisks are marked.

The antidepressant effect of the compound was verified through animal experiments, and the experimental data are shown in Table 2 and Figure 18.

Table 2. Effects of low, medium and high doses of PL171 administered intragastrically on the time of forced swimming immobility in mice

("*" means P＜0.05, "**" means P＜0.01, compared with the vehicle group)

6. Experimental results

The results show that the low, medium and high doses of PL171 can significantly shorten the immobile time of forced swimming in mice, suggesting that they have significant antidepressant activity. Among them, the antidepressant effect of high-dose PL171 is more significant.

Example 15: Evaluation of antidepressant efficacy of PL171 in mice for a long period of time

1. Forced swimming experiment

1. Reagents and medicines: the same as in Example 13

2. Animals: same as Example 13

3. Method: Divide healthy C57BL/6J mice into 5 groups randomly, 10 mice in each group, which are vehicle group (three distilled water); control group (fluoxetine hydrochloride 20.0mg/kg); PL171 high and medium , Low-dose group (50.0, 15.0, 5.0 mg/kg), intragastric administration, once a day, for 7 consecutive days. During the experiment, the animals were free to eat and drink. 24 hours after the last administration, the mice were placed in a container with a diameter of about 18 cm, a water depth of 18 cm, and a water temperature of 25°C. The swimming time of the mice was 6 minutes, and the mice were floating motionless within 4 minutes. (Ie, the mouse stops struggling in the water, or the animal is floating, with only small limbs moving to keep the head floating on the water).

4. Data on forced swimming immobility time

table 3

5 Conclusion

The three doses of PL171 have significant antidepressant effects. Because of forced swimming after 24 hours of administration, fluoxetine takes 2-3 weeks to take effect, which also indicates that PL171 may have a rapid antidepressant effect.

Second, the tail suspension experiment

1. Reagents and medicines: the same as in Example 13

2. Animals: After 7 consecutive days after the forced swimming test in C57BL / 6J mice

3. Methods: After 7 consecutive days, healthy after forced swimming test C57BL / 6J mice continued intragastric administration once / day for 14 days. During the experiment, the animals were free to eat and drink. 24 hours after the last administration, the tail of the mouse was fixed with a clip about 1 cm from the end, and the mouse was hung upside down on a crossbar about 15 cm from the ground. The animal struggled to overcome the abnormal posture, but after a period of activity, Intermittent immobility appeared, showing a state of despair, hanging for 6 minutes, and accumulating the immobility time within 4 minutes after each group. This time, also known as the tail suspension time, is also an indicator for determining the degree of depression known in the art. The shorter the time, the higher the activity of the mouse and the better the antidepressant effect.

4. The data of the hanging tail immobility time

Table 4

5 Conclusion

The three doses of compound PL171 can significantly reduce the immobility time of mice, suggesting that the antidepressant effect is significant, showing an obvious dose-effect relationship. After 14 days of continuous administration of the positive drug fluoxetine, the mice also showed significant antidepressant effects. Because fluoxetine has no obvious effect after 7 days of administration, while PL171 has an antidepressant effect after 7 days, it indicates that PL171 has a significant antidepressant effect, and the antidepressant has a faster onset time, and the effective dose is also lower than fluoxetine. . Combined with previous studies on SIRT3 as a key molecule for PL171, it is likely that the preventive and therapeutic effects of depression can also be achieved by increasing the activity/level of SIRT3.

In summary, the results of Examples 13-14 show that PL-171 can quickly and continuously exert anti-depressant effects in a single dose and multiple repeated doses at a smaller dose than the positive control. Prevention, relief and treatment of symptoms and symptoms.

Example 16: Test results of the effect of PL171 on the ability of reaction inhibition

1. Introduction to the Stop-signal task model

The Stop-signal task model is shown in Figures 15A and 15B, and its further details are described in the reference published in Acta Pharmacologica Sinica volume on December 21, 2017, "Prefrontal AMPA receptors are involved in the effect of" methylphenidate on response inhibition in rats", which is incorporated herein by reference in its entirety. In short, provide a chamber with 3 poking ports, the middle port is used as a reward port (reward), each correct test will provide a drop of water, use an infrared detector to monitor the entry into any of the three ports Poke your nose. The elapsed time from leaving the initial port to poking the nose into the action port is defined as Go response time (Go RT).

The following behavioral evaluation indicators were measured:

The behavior test includes a session, a session has 320 trials, and the 21st-320th trial is used for parameter calculation. Divided into 3 blocks, each block contains 100 trials, of which 80 go trials and 20 stop trials.

(1) Stop-signal reaction time (SSRT): indicates the ability of reaction inhibition and internal decision-making power. The smaller the SSRT value, the better the reaction inhibition ability, and vice versa.

Each correct go trial can calculate a Go response time (Go RT), arrange the Go RT of each block from small to large, take the value of the nth Go RT, and then subtract the stop signal of 20 stop trials The average value of the stop-signal delay (SSD) that appears. The final SSRT is the average of 3 SSRTs calculated by 3 blocks.

SSRT=GoRT(n)-SSD average value, where n=Go trial number x (1-stop operation correct rate).

(2) Stop accuracy (Stop accuracy): indicates the ability to inhibit reaction and disciplinary reaction. The change of the correct rate of stopping operation can reflect the change of the reaction inhibition ability. If it becomes larger, it indicates that the reaction inhibition ability is enhanced.

Go trial reaction time (GoRT) = the time when the animal probes the nose into the behavioral port (ms)-the time when the nose is withdrawn from the initial port (ms).

(3) Go reaction time: indicates exercise reaction ability and reward reaction.

(4) Go trial operation accuracy (Go accuracy): indicates memory ability. The Go operation accuracy rate can reflect the application of the animal's operation rules for behavioral tasks. If the accuracy of the Go operation becomes smaller, it indicates that it affects the use of familiar operating rules by animals, and involves changes in memory ability.

Go correct rate = number of Go trials with correct operations/240 (the number of Go trials with 3 blocks is 240).

Stop accuracy rate = the number of stop trials with correct operations/60 (the number of stop trials for 3 blocks is 60).

2. Experimental design

1. Drug preparation and use:

Preparation: Use ddH ₂ O to make a 1.0 mg/ml solution of PL171 and store at 4°C.

Usage: 10mg/kg dose is given at 1.0ml/100g, that is, 1ml of medicine is given to 100g body weight. At a dose of 5mg/kg, dilute the drug twice before use.

An equal volume of ddH ₂ O was used as a control, with 1.0ml/100g body weight.

Administration: 1.0 mg/ml PL171 solution was fed.

2. Animals:

Male Sprague-Dawley rats (160-180g) were purchased from SLACC (Shanghai, China). All rats were grouped and housed in a light/dark cycle of 12:12 (lights on at 8:00 in the morning). Food and water are freely available. The rats are weighed daily to ensure that about 95% of their original body weight is maintained. All experimental procedures were carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health" (NIH Publication No. 80-23, 1996), and were approved and monitored by the animal experiment ethics committees of the institutes. College (Shanghai, China).

3. Dosing schedule (see Figure 15C)

Baseline: Rats start to do the drug after the test is relatively stable for two consecutive days. The average of the two days' test is used as the baseline. Test1: Test after 2 consecutive administrations (each dose is 10 mg/kg), and conduct a behavioral test 3 hours after the second administration. Test2: Test after 5 consecutive administrations (the first 2 doses are 10 mg/kg, the last 3 doses are 5 mg/kg), and the behavioral test is performed 3 hours after the 5th administration. Test3: A test 48 hours after the 5th dose to compare whether the results of Test1 and Test2 are caused by drug effects or repeated behavioral tests.

3. Experimental results

Experimental result 1: The effect of drugs on Stop trial operation

The results in Fig. 16 show that the reaction inhibition ability of the rats was significantly improved after taking the medicine. As shown in Fig. 16A, the signal stop reaction time (SSRT) of the rats was significantly shortened after taking the medicine, and there was no change in the drinking water control group. 48 hours after stopping the medication, the rat's SSRT no longer decreased, indicating that the changes in SSRT in Test1 and Test2 were the effect of the drug.

As shown in Figure 16B, the correct rate of Stop trial operation in rats was significantly improved after taking the drug, and the drinking water control group had no effect (**p<0.01, *p<0.05, Wilcoxon rank sum test).

Experimental result 2: The effect of drugs on Go trial operation

The results in Figure 17 show that the rats' exercise response ability and the use of behavioral task rules are not affected after taking the drug. As shown in Fig. 17A, the Go reaction time (GoRT) of the rats did not change after taking the drug, indicating that the drug had no effect on the exercise response ability of the rats. As shown in Figure 17B, the Go trial accuracy of rats after taking the drug is not affected, indicating that the drug does not affect the rat's use of behavioral rules.

In summary, the above results indicate that PL171 significantly reduces the animal’s stop signal response time and increases the accuracy of Stop (Figure 16); PL171 significantly improves and improves the ability of rats to inhibit response, that is, improves and improves the cognitive ability of rats .

Those skilled in the art will further realize that the present invention can be implemented in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the present invention only discloses exemplary embodiments thereof, it should be understood that other changes are considered to be within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Instead, reference should be made to the appended claims to indicate the scope and content of the invention.

Claims (24)

1. A method for preparing the compound N-(β-L-rhamnopyranosyl) ferulic acid amide of formula I, the method comprises the following steps:

   1) Compound 2 is reacted with compound 3 in the presence of a base to obtain compound 1;

   2) Compound 1 undergoes a deprotection reaction under the conditions of a deprotecting agent to obtain compound I;

   

   The structural formula of compound 3 is as follows:

   

   in,

   P is selected from All, Boc, TMS, TES, TBS, TIPS, TBDPS, THP, MOM, MTM, MEM, BOM, SEM, EE, Bn, PMB, Cbz, DMB and Tr;

   X is selected from Cl and Br.
2. The method of claim 1, wherein:

   The reaction temperature in the step 1) is -25°C-100°C, and the reaction solvent is selected from methanol, ethanol, propanol, isopropanol, tert-butanol, n-butanol, pyridine, dichloromethane, tetrahydrofuran, 2-methyl Tetrahydrofuran (2-MeTHF), water or any combination thereof; and/or

   The reaction temperature of the step 2) is -5°C-60°C, and the reaction solvent is methanol, ethanol, propanol, isopropanol, tert-butanol, n-butanol, acetonitrile, 1,4-dioxane, tetrahydrofuran , Methylene chloride, or any combination thereof.
3. The method of claim 1 or 2, wherein:

   In step 1), the base is selected from one or more of inorganic bases or organic bases; the molar ratio of compound 2 to base is 1:1-7; the molar ratio of compound 2 to compound 3 is 0.8-3 : 1-4; and/or

   In step 2), the molar ratio of compound 1 to the deprotection agent is 1:0.1-4.
4. The method according to claim 1, further comprising the step of reacting the rhamnose compound with an ammonia source to obtain compound 2.
5. The method according to claim 4, wherein the reaction temperature of the reaction is 15°C-100°C, the reaction time is 0.5-60h, and the reaction solvent is an alcohol solvent; the molar ratio of the rhamnose compound and the ammonia source The ratio is 1:1-10.
6. The method according to claim 1, further comprising subjecting compound 5 to a hydroxyl protection reaction in an organic solvent to obtain compound 6, subjecting compound 6 to an alkaline hydrolysis reaction to obtain compound 7, and subjecting compound 7 to a halogenation reaction to obtain compound 3. A step of,

   
7. The method of claim 6, wherein

   The compound 5 undergoes a hydroxyl protection reaction with a hydroxyl protecting reagent under acid binding agent conditions to obtain compound 6; the compound 6 undergoes alkaline hydrolysis under alkaline conditions to obtain compound 7;

   Compound 7 is reacted with halogenating reagent to obtain compound 3.
8. The method of claim 7, wherein

   The reaction temperature in the hydroxyl protection reaction is -5-70°C, and the reaction time of the hydroxyl protection reaction is 1-24 h; the molar ratio of the compound 5 to the acid binding agent is 1:1-6, and the compound 5 The molar ratio with the hydroxyl protecting reagent is 1:1-5;

   The alkaline hydrolysis reaction solvent is tetrahydrofuran aqueous solution, the alkaline hydrolysis reaction temperature is room temperature, and the reaction time is 1-10h; the molar ratio of the compound 6 to the base is 1:0.1-1;

   The reaction temperature in the halogenation reaction is 10-60°C, the reaction time of the halogenation reaction is 1-10h, and the halogenation reaction solvent is selected from dichloromethane, acetonitrile or a combination thereof; the compound 7 and the halogenation reagent The molar ratio is 1:1-5.
9. Compound of formula 1:

   

   Wherein, P is selected from All, Boc, TMS, TES, TBS, TIPS, TBDPS, THP, MOM, MTM, MEM, BOM, SEM, EE, Bn, PMB, Cbz, DMB and Tr.
10. Use of N-(β-L-rhamnopyranosyl) ferulic acid amide and a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, alleviating or treating abnormal mitochondrial function in cells of a subject.
11. The use according to claim 10, wherein the mitochondrial dysfunction is an Aβ protein-induced mitochondrial dysfunction.
12. The use according to claim 11, wherein the Aβ protein is an oligomer of Aβ42 peptide (Aβ42O).
13. The use according to any one of claims 10-12, wherein the mitochondrial dysfunction includes an increase in the level of protein acetylation in the mitochondria, an increase in the level of reactive oxygen species, a decrease in membrane potential and/or a decrease in oxygen consumption.
14. The use according to any one of claims 11-13, wherein the mitochondria are mitochondria in nerve cells.
15. Use of N-(β-L-rhamnopyranosyl) ferulic acid amide and a pharmaceutically acceptable salt thereof in the preparation of a medicament for increasing the activity or level of SIRT3 in a subject.
16. The use according to claim 15, wherein the drug also enhances the activity or level of AMPK and/or PGC-1.
17. N-(β-L-rhamnanopyranosyl) ferulic acid amide and its pharmaceutically acceptable salts are prepared for reducing mitochondrial protein acetylation, oxidative stress levels or reactive oxygen levels in the cells of subjects Use in medicines.
18. The use according to claim 17, wherein the cell is a nerve cell.
19. N-(β-L-rhamnanopyranosyl) ferulic acid amide and its pharmaceutically acceptable salt are prepared for the prevention, alleviation or treatment of depression in subjects in short-term and long-term periods the use of.
20. The use according to claim 19, wherein the medicament rapidly and continuously prevents, relieves or treats depression in a short-term and a long-term period.
21. N-(β-L-rhamnopyranosyl) ferulic acid amide and its pharmaceutically acceptable salt are used in the preparation of drugs for preventing cognitive impairment, improving or enhancing cognitive ability of subjects In the use.
22. The use according to claim 21, wherein the cognitive ability is response inhibition ability and/or memory ability.
23. Use of N-(β-L-rhamnopyranosyl) ferulic acid amide and a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting or delaying the senescence of a subject.
24. The use according to claim 23, wherein the senescence is related to SIRT3.

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