CN115651062B - Derivatives of maleic acid, preparation method and application thereof - Google Patents
Derivatives of maleic acid, preparation method and application thereof Download PDFInfo
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- CN115651062B CN115651062B CN202211264173.5A CN202211264173A CN115651062B CN 115651062 B CN115651062 B CN 115651062B CN 202211264173 A CN202211264173 A CN 202211264173A CN 115651062 B CN115651062 B CN 115651062B
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- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention belongs to the technical field of medicines, and particularly relates to a maleic acid derivative, and a preparation method and application thereof. The invention provides a maleic acid derivative, which has a structure shown in a formula 1. The animal model experiment shows that: after model mice are treated by the maleic acid derivative with the structure shown in the formula 1: in the immunohistochemical experiment, the average optical density of the TH positive region of the substantia nigra compact part of the treated mice is obviously increased; the activity of the black matrix SOD and GSH-px of the treated mice is obviously increased, and the MDA content is obviously reduced; the black sphingomyelin and ceramide content of the treated mice are obviously reduced. The overall activity result shows that the loganin acid derivative provided by the invention can protect or repair the damage of the nervous system and has good treatment effect on the degenerative diseases or symptoms of the parkinsonism.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a maleic acid derivative, and a preparation method and application thereof.
Background
Neurodegenerative diseases are pathologically represented by progressive, irreversible dysfunction and neuronal loss, and behaviorally represented by a decline or even loss of learning and memory abilities, and are the main features of diseases such as Alzheimer's Disease (AD) and Parkinson's Disease (PD). Wherein, parkinson is one of the common clinical diseases, seriously affects the health of middle-aged and elderly people, and mainly clinically presents with resting tremor, bradykinesia, myotonia, posture gait disorder and the like. Parkinson's disease is a neurodegenerative disease next to alzheimer's disease, with ambiguous etiology.
At present, the treatment of the parkinsonism has become an important direction of medical research, and the treatment medicine is mainly western medicine, wherein the levodopa preparation is one of the most effective medicines, but the side effect is obvious, and the larger the dosage is, the larger the side effect is. There is thus an urgent need to develop effective medicaments for the treatment of parkinson's disease.
Disclosure of Invention
The invention aims to provide a derivative of maleic acid, a preparation method and application thereof, and the derivative of maleic acid provided by the invention can protect or repair damage of a nervous system and has good treatment effect on degenerative diseases or symptoms of the parkinsonism.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a maleic acid derivative, which has a structure shown in a formula 1:
The invention provides a preparation method of the loganin acid derivative, which comprises the following steps:
Mixing loganin acid, a compound with a structure shown in a formula 2, a coupling reagent and an organic solvent for coupling reaction to obtain a compound with a structure shown in a formula 3;
And in a hydrogen atmosphere, mixing the compound with the structure shown in the formula 3, a palladium-carbon catalyst and an organic solvent for reduction and deprotection reaction to obtain the derivative of the maleic acid with the structure shown in the formula 1.
Preferably, the preparation method of the compound with the structure shown in the formula 2 comprises the following steps:
Mixing a compound with a structure shown in a formula 4, a compound with a structure shown in a formula 5, a condensation reagent, a dehydrating agent, an organic base reagent and an organic solvent for carrying out an amide condensation reaction to obtain a compound with a structure shown in a formula 6;
And mixing the compound with the structure shown in the formula 6 with an organic solution of hydrogen chloride to perform amino deprotection reaction to obtain the compound with the structure shown in the formula 2.
Preferably, the preparation method of the compound with the structure shown in the formula 4 comprises the following steps:
mixing a compound with a structure shown in a formula 7, a compound with a structure shown in a formula 8, a condensation reagent, a dehydrating agent, an organic alkali reagent and an organic solvent for condensation reaction to obtain a compound with a structure shown in a formula 9;
and mixing the compound with the structure shown in the formula 9, an organic solvent and an inorganic alkali solution for hydrolysis reaction to obtain the compound with the structure shown in the formula 4.
Preferably, the preparation method of the compound with the structure shown in the formula 5 comprises the following steps:
mixing a compound with a structure shown in a formula 10, a compound with a structure shown in a formula 11, a condensation reagent, a dehydrating agent, an organic alkali reagent and an organic solvent for condensation reaction to obtain a compound with a structure shown in a formula 12;
And mixing the compound with the structure shown in the formula 12 with an organic solution of hydrogen chloride to perform amino deprotection reaction to obtain the compound with the structure shown in the formula 5.
Preferably, the molar ratio of Ma Qiansuan to the compound of the structure shown in formula 2 is 1 (1.9-2.2).
Preferably, the molar ratio of the compound of the structure represented by formula 4 to the compound of the structure represented by formula 5 is 1 (1.1 to 1.3).
Preferably, the mass ratio of the compound of the structure represented by formula 7 to the compound of the structure represented by formula 8 is 1 (1.1 to 1.3).
Preferably, the molar ratio of the compound of the structure represented by formula 10 to the compound of the structure represented by formula 11 is 1 (1.1 to 1.3).
The invention provides an application of the loganin acid derivative or the loganin acid derivative prepared by the preparation method of the technical scheme in preparing medicines for treating and/or preventing parkinsonism degenerative diseases or symptoms.
The invention provides a maleic acid derivative, which has a structure shown in a formula 1. The derivative of loganin provided by the invention couples loganin with a short peptide, wherein the short peptide is a peptide sequence with an arginine-glycine-aspartic acid (RGD) motif, and the short peptide shows strong affinity to integrins (integrins), especially alpha Vβ3 integrins. Integrins are transmembrane glycoproteins that are involved in the interaction of cells with other cells or with the extracellular matrix. Integrins are actively expressed on the surface of vascular endothelial cells and play an important role in angiogenesis, leukocyte migration and tumor metastasis. This makes integrins suitable targets for the treatment of various inflammatory diseases and cancers. Therefore, the invention adopts the RGD sequence peptide coupling to realize targeting effect on the derivative of the loganin obtained after the loganin and the short peptide are coupled. The animal model experiment shows that: after model mice are treated by the maleic acid derivative with the structure shown in the formula 1: in the open field experiment, the total moving distance, the crossing frequency, the center moving distance and the standing frequency of the treated mice are obviously increased; in the pole-climbing experiment, the pole-climbing time of the treated mice is obviously reduced; in the immunohistochemical experiment, the average optical density of the TH positive region of the substantia nigra compact part of the treated mice is obviously increased; in TUNEL experiments, the number of treated mice substantia nigra TUNEL positive cells was significantly reduced; in the immunofluorescence experiment, the average fluorescence intensity of the black matter SYN positive region of the treated mice is obviously reduced, and the average fluorescence intensity of the black matter PSD-95 positive region of the treated mice is obviously reduced; the black matrix DA, DOPAC, HVA and 5-HT content of the treated mice are obviously increased, and the IL-1 beta and TNF-alpha content is obviously reduced; the activity of the black matrix SOD and GSH-px of the treated mice is obviously increased, and the MDA content is obviously reduced; the black sphingomyelin and ceramide content of the treated mice are obviously reduced. The overall activity result shows that the loganin acid derivative provided by the invention can protect or repair the damage of the nervous system and has good treatment effect on the degenerative diseases or symptoms of the parkinsonism.
The invention provides a preparation method of the loganin acid derivative, which comprises the following steps: mixing loganin acid, a compound with a structure shown in a formula 2, a coupling reagent and an organic solvent for coupling reaction to obtain a compound with a structure shown in a formula 3; and in a hydrogen atmosphere, mixing the compound with the structure shown in the formula 3, a palladium-carbon catalyst and an organic solvent for reduction and deprotection reaction to obtain the derivative of the maleic acid with the structure shown in the formula 1. The preparation method provided by the invention uses the coupling of the loganin acid and the full-protection tetrapeptide and then hydrogenates to successfully obtain the tetrapeptide modified loganin acid composed of arginine-glycine-aspartic acid-valine, can effectively protect or repair the damage of the nervous system, and has good treatment effect on the degenerative diseases or symptoms of the parkinsonism nervous system. The preparation method is simple and is suitable for industrial production.
Drawings
FIG. 1 is a synthetic route diagram provided by an embodiment of the present invention;
FIG. 2 is a mass spectrum of a compound of the structure shown in formula 6 prepared in the example of the present invention;
FIG. 3 is a mass spectrum of a derivative of loganin prepared in an embodiment of the invention;
FIG. 4 is a diagram showing a hydrogen nuclear magnetic spectrum of a compound having a structure shown in formula 6 prepared in the example of the present invention;
FIG. 5 is a hydrogen nuclear magnetic spectrum of a derivative of logenic acid prepared according to an embodiment of the present invention;
FIG. 6 is an ultraviolet spectrum of a derivative of loganin prepared in an embodiment of the invention;
FIG. 7 shows the effect of the derivatives of loganin prepared in the examples of the invention on PD mouse behavior;
FIG. 8 shows the effect of the derivatives of loganin prepared in the examples of the invention on the TH expression of SN tissue of PD mice;
FIG. 9 is a graph showing SN results of PD mice with respect to derivatives of loganin prepared in the example of the invention;
FIG. 10 shows the effect of the derivatives of loganin prepared in the examples of the invention on the expression of SYN in SN tissue of PD mice;
FIG. 11 shows the effect of the loganin derivative prepared in the example of the invention on PSD-95 expression of SN tissue of PD mice;
FIG. 12 shows the effect of the derivatives of loganin prepared in the examples of the invention on SN tissue DA, DOPAC, HVA, 5-HT, IL-1. Beta. And TNF-. Alpha.expression in PD mice;
FIG. 13 shows the effect of the derivatives of loganin prepared in the examples of the invention on SOD, MDA, GSH-px expression in SN tissue of PD mice;
FIG. 14 shows the effect of the derivatives of loganin prepared in the examples of the invention on SN tissue sphingomyelin and ceramide in PD mice.
Detailed Description
The invention provides a maleic acid derivative, which has a structure shown in a formula 1:
The invention provides a preparation method of the loganin acid derivative, which comprises the following steps:
Mixing loganin acid, a compound with a structure shown in a formula 2, a coupling reagent and an organic solvent for coupling reaction to obtain a compound with a structure shown in a formula 3;
And in a hydrogen atmosphere, mixing the compound with the structure shown in the formula 3, a palladium-carbon catalyst and an organic solvent for reduction and deprotection reaction to obtain the derivative of the maleic acid with the structure shown in the formula 1.
In the present invention, all preparation materials/components are commercially available products well known to those skilled in the art unless specified otherwise.
The invention mixes the loganin acid, the compound with the structure shown in the formula 2, the coupling reagent and the organic solvent for coupling reaction to obtain the compound with the structure shown in the formula 3.
In the invention, the structural formula of the monic acid is shown as formula 13:
in the present invention, the preparation method of the compound of the structure represented by formula 2 preferably comprises the steps of:
Mixing a compound with a structure shown in a formula 4, a compound with a structure shown in a formula 5, a condensation reagent, a dehydrating agent, an organic base reagent and an organic solvent for carrying out an amide condensation reaction to obtain a compound with a structure shown in a formula 6;
And mixing the compound with the structure shown in the formula 6 with an organic solution of hydrogen chloride to perform amino deprotection reaction to obtain the compound with the structure shown in the formula 2.
The invention mixes the compound with the structure shown in the formula 4, the compound with the structure shown in the formula 5, the condensation reagent, the dehydrating agent, the organic alkali reagent and the organic solvent for amide condensation reaction to obtain the compound with the structure shown in the formula 6.
In the present invention, the preparation method of the compound of the structure represented by formula 4 preferably comprises the steps of:
mixing a compound with a structure shown in a formula 7, a compound with a structure shown in a formula 8, a condensation reagent, a dehydrating agent, an organic alkali reagent and an organic solvent for condensation reaction to obtain a compound with a structure shown in a formula 9;
and mixing the compound with the structure shown in the formula 9, an organic solvent and an inorganic alkali solution for hydrolysis reaction to obtain the compound with the structure shown in the formula 4.
In the present invention, a compound having a structure represented by formula 7, a compound having a structure represented by formula 8, a condensation reagent, a dehydrating agent, an organic base reagent, and an organic solvent are mixed to perform a condensation reaction (hereinafter referred to as a first condensation reaction), thereby obtaining a compound having a structure represented by formula 9.
In the present invention, the first condensation reaction is performed by: the condensing agent is particularly preferably 1-hydroxybenzotriazole (HOBt). The dehydrating agent is preferably Dicyclohexylcarbodiimide (DCC). The organic base reagent is preferably N-methylmorpholine. The organic solvent is particularly preferably anhydrous tetrahydrofuran.
In the present invention, the first condensation reaction is performed by: the molar ratio of the compound of the structure represented by formula 7 to the compound of the structure represented by formula 8 is preferably 1 (1.1 to 1.3), more preferably 1:1.2. The molar ratio of the compound of the structure represented by formula 7 to the condensing agent is preferably 1:1.2. The molar ratio of the compound of the structure represented by formula 7 to the dehydrating agent is preferably 1:1.1. The amount of the organic base reagent is preferably such that the pH of a mixed solution obtained by mixing the compound having the structure represented by formula 7, the compound having the structure represented by formula 8, the condensation reagent, the dehydrating agent and the organic solvent is adjusted to 8 to 9. The invention has no special requirement on the dosage of the organic solvent, and ensures that the first condensation reaction is carried out smoothly.
In the present invention, the first condensation reaction is performed by: the mixing preferably comprises the steps of: mixing a compound with a structure shown in a formula 7 with an organic solvent to obtain a compound solution with a structure shown in the formula 7; stirring and mixing a compound solution with a structure shown in a formula 7, a condensation reagent and a dehydrating agent for 20-30 min under the ice water bath condition to obtain a first mixed solution; mixing the first mixed solution with a compound with a structure shown in a formula 8 to obtain a second mixed solution; and (3) dropwise adding the organic alkali reagent into the second mixed solution, adjusting the pH value of the second mixed solution to 8-9, and removing the ice water bath.
In the present invention, the temperature of the first condensation reaction is preferably room temperature. The time of the first condensation reaction is preferably overnight. The first condensation reaction is carried out with stirring. The first condensation reaction is preferably detected by TLC using preferably CH 2Cl2/CH3 OH, v: v=20/1.
In the present invention, the first condensation reaction is performed to obtain a first condensation reaction solution, and the present invention preferably performs a post-treatment on the first condensation reaction solution to obtain a compound having a structure represented by formula 9. In the present invention, the post-treatment preferably includes: concentrating the first condensation reaction liquid to obtain a residue; mixing the residue with ethyl acetate, and then carrying out solid-liquid separation to obtain ethyl acetate filtrate; and washing, drying and removing the solvent from the ethyl acetate filtrate in sequence to obtain the compound with the structure shown in the formula 9. In the present invention, the concentration is preferably concentration under reduced pressure. The solid-liquid separation is preferably filtration, and the present invention preferably removes impurity impurities generated after DCC participates in the reaction by solid-liquid separation. The washing is preferably: sequentially carrying out saturated NaHCO 3 aqueous solution washing, saturated NaCl aqueous solution washing and saturated KHSO 4 aqueous solution washing; each solution was washed 3 times. In the present invention, the drying is preferably carried out using anhydrous sodium sulfate for 2 hours or more. In the present invention, the specific embodiment of the solvent removal is preferably concentration under reduced pressure.
After the compound with the structure shown in the formula 9 is obtained, the compound with the structure shown in the formula 9, an organic solvent and an inorganic alkali solution are mixed for hydrolysis reaction to obtain the compound with the structure shown in the formula 4.
In the present invention, the organic solvent is preferably methanol in the hydrolysis reaction. The organic alkali solution is preferably an aqueous NaOH solution. The molar concentration of the inorganic alkali solution is preferably 2mol/L. The invention has no special requirement on the dosage of the organic solvent, and can completely dissolve the compound with the structure shown in the formula 9. The amount of the inorganic alkali solution is preferably such that the pH of the organic solution of the compound of formula 9 is adjusted to 13 to 14.
In the present invention, in the hydrolysis reaction, the mixing is preferably: dissolving a compound with a structure shown in a formula 9 in an anhydrous methanol organic solvent to obtain an organic solution of the compound with the structure shown in the formula 9; and mixing the inorganic alkali solution and the organic solution of the compound with the structure shown in the formula 9 under the ice water bath condition, and adjusting the pH value of the organic solution of the compound with the structure shown in the formula 9 to 13-14.
In the present invention, the hydrolysis reaction is preferably carried out with stirring under ice-water bath conditions. In the present invention, the time of the hydrolysis reaction is preferably 3.5 to 4 hours. The hydrolysis reaction is preferably detected by TLC using a developing reagent preferably CH 2Cl2/CH3 OH, v: v=20/1.
In the present invention, the hydrolysis reaction is performed to obtain a hydrolysis reaction solution, and the present invention preferably performs post-treatment on the hydrolysis reaction solution to obtain a compound having a structure represented by formula 4. In the present invention, the post-treatment preferably includes: mixing the hydrolysis reaction liquid with saturated KHSO 4 under the condition of ice water bath, and regulating the pH value of the reaction liquid to 7; removing the organic solvent by hydrolysis reaction with pH value of 7 to obtain residue; re-dissolving the residue with pure water under the ice water bath condition, regulating the pH value of the residue to 2 with a saturated KHSO 4 solution, and re-dissolving with an ethyl acetate solution; extracting the residue with pH value of 2 to obtain an extracted organic phase; washing, drying and removing the extraction solvent are sequentially carried out on the extracted organic phase, so that the compound with the structure shown in the formula 4 is obtained. The specific embodiment for removing the organic solvent is preferably concentration under reduced pressure. The extraction agent is preferably ethyl acetate. The number of extractions is preferably 3 and the organic phases are combined for drying. The drying is preferably carried out overnight with anhydrous sodium sulfate. The specific embodiment for removing the extraction solvent is preferably concentration under reduced pressure.
In the present invention, the preparation method of the compound of the structure represented by formula 5 preferably comprises the steps of:
mixing a compound with a structure shown in a formula 10, a compound with a structure shown in a formula 11, a condensation reagent, a dehydrating agent, an organic alkali reagent and an organic solvent for condensation reaction to obtain a compound with a structure shown in a formula 12;
And mixing the compound with the structure shown in the formula 12 with an organic solution of hydrogen chloride to perform amino deprotection reaction to obtain the compound with the structure shown in the formula 5.
The invention mixes the compound with the structure shown in the formula 10, the compound with the structure shown in the formula 11, the condensation reagent, the dehydrating agent, the organic alkali reagent and the organic solvent for condensation reaction (second condensation reaction) to obtain the compound with the structure shown in the formula 12.
In the present invention, the second condensation reaction is performed by: the condensing agent is particularly preferably 1-hydroxybenzotriazole (HOBt). The dehydrating agent is preferably Dicyclohexylcarbodiimide (DCC). The organic base reagent is preferably N-methylmorpholine. The organic solvent is particularly preferably anhydrous tetrahydrofuran.
In the present invention, the second condensation reaction is performed by: the molar ratio of the compound of the structure represented by formula 10 to the compound of the structure represented by formula 11 is preferably 1 (1.1 to 1.3), more preferably 1:1.2. The molar ratio of the compound of the structure represented by formula 10 to the condensing agent is preferably 1:1.2. The molar ratio of the compound of the structure represented by formula 10 to the dehydrating agent is preferably 1:1.1. The amount of the organic base reagent is preferably such that the pH of a mixed solution obtained by mixing the compound having the structure represented by formula 10, the compound having the structure represented by formula 11, the condensing reagent, the dehydrating agent and the organic solvent is adjusted to 8 to 9. The invention has no special requirement on the dosage of the organic solvent, and ensures that the second condensation reaction is carried out smoothly.
In the present invention, the second condensation reaction is performed by: the mixing preferably comprises the steps of: mixing a compound with a structure shown in a formula 10 with an organic solvent to obtain a compound solution with a structure shown in the formula 10; stirring and mixing a compound solution with a structure shown in a formula 10, a condensation reagent and a dehydrating agent for 20-30 min under the ice water bath condition to obtain a first mixed solution; mixing the first mixed solution with a compound with a structure shown in a formula 11 to obtain a second mixed solution; and (3) dropwise adding the organic alkali reagent into the second mixed solution, adjusting the pH value of the second mixed solution to 8-9, and removing the ice water bath.
In the present invention, the temperature of the second condensation reaction is preferably room temperature. The time of the second condensation reaction is preferably overnight. The second condensation reaction is carried out with stirring. The second condensation reaction is preferably detected by TLC using a developing reagent preferably CH 2Cl2/CH3 OH, v: v=20/1.
In the present invention, the second condensation reaction is performed to obtain a second condensation reaction solution, and the present invention preferably performs a post-treatment on the second condensation reaction solution to obtain a compound having a structure represented by formula 12. In the present invention, the post-treatment preferably includes: concentrating the second condensation reaction liquid to obtain a residue; mixing and dissolving the residues and ethyl acetate, then carrying out solid-liquid separation, and removing DCU generated after DCC participates in the reaction to obtain ethyl acetate filtrate; and washing, drying and removing the solvent from the ethyl acetate filtrate in sequence to obtain the compound with the structure shown in the formula 12. In the present invention, the concentration is preferably concentration under reduced pressure. The solid-liquid separation is preferably filtration, and the impurity is preferably removed by solid-liquid separation in the present invention. The washing is preferably: sequentially carrying out saturated NaHCO 3 aqueous solution washing, saturated NaCl aqueous solution washing and saturated KHSO 4 aqueous solution washing; each solution is preferably washed 3 times. In the present invention, the drying is preferably performed overnight with anhydrous sodium sulfate. In the present invention, the specific embodiment of the solvent removal is preferably concentration under reduced pressure.
After obtaining the compound of the structure represented by formula 12, the present invention mixes the compound of the structure represented by formula 12 with an organic solution of hydrogen chloride to perform an amino deprotection reaction (hereinafter referred to as a first amino deprotection reaction) to obtain the compound of the structure represented by formula 5.
In the present invention, in the first amino deprotection reaction, the organic solution of hydrogen chloride is preferably an ethyl acetate solution of hydrogen chloride. The molar concentration of hydrogen chloride in the organic solvent of hydrogen chloride is preferably 4mol/L. The ratio of the mass of the compound of the structure represented by formula 12 to the volume of the organic solvent of hydrogen chloride is preferably 1g:10mL.
In the present invention, the temperature of the first amino group deprotection reaction is preferably room temperature, the time of the first amino group deprotection reaction is preferably 4 hours, and the first amino group deprotection reaction is preferably carried out under stirring. The first amino deprotection reaction is preferably detected by TLC using a developing reagent preferably CH 2Cl2/CH3 OH, v: v=20/1.
In the present invention, the first deprotection reaction of the first amino group is carried out to obtain a first deprotection reaction solution, and the present invention preferably carries out a post-treatment on the first deprotection reaction solution to obtain a compound having a structure represented by formula 5. In the present invention, the post-treatment preferably includes: the first deprotected reaction solution was first pumped out under reduced pressure with a water pump, and then washed 3 times with 20ml of anhydrous ethyl acetate. In the present invention, the condensing agent is particularly preferably 1-hydroxybenzotriazole (HOBt) in the amide condensation reaction. The dehydrating agent is preferably Dicyclohexylcarbodiimide (DCC). The organic base reagent is preferably N-methylmorpholine. The organic solvent is particularly preferably anhydrous tetrahydrofuran.
In the present invention, the amide condensation reaction is performed by: the molar ratio of the compound of the structure represented by formula 4 to the compound of the structure represented by formula 5 is preferably 1 (1.1 to 1.3), more preferably 1:1.2. The molar ratio of the compound of the structure of formula 4 to the condensing agent is preferably 1:1.2. The molar ratio of the compound of the structure represented by formula 4 to the dehydrating agent is preferably 1:1.1. The amount of the organic base reagent is preferably such that the pH of a mixed solution obtained by mixing the compound having the structure represented by formula 4, the compound having the structure represented by formula 5, the condensation reagent, the dehydrating agent and the organic solvent is adjusted to 8 to 9. The invention has no special requirement on the dosage of the organic solvent, and ensures that the amide condensation reaction is carried out smoothly.
In the present invention, the amide condensation reaction is performed by: the mixing preferably comprises the steps of: mixing a compound with a structure shown in a formula 4 with an organic solvent to obtain a compound solution with a structure shown in the formula 4; stirring and mixing a compound solution with a structure shown in a formula 4, a condensation reagent and a dehydrating agent for 20-30 min under the ice water bath condition to obtain a first mixed solution; mixing the first mixed solution with a compound with a structure shown in a formula 5 to obtain a second mixed solution; and (3) dropwise adding the organic alkali reagent into the second mixed solution, adjusting the pH value of the second mixed solution to 8-9, and removing the ice water bath.
In the present invention, the temperature of the amide condensation reaction is preferably room temperature. The amide condensation reaction is preferably carried out overnight. The amide condensation reaction is carried out under stirring. The amide condensation reaction is preferably detected by TLC using a developing reagent preferably CH 2Cl2/CH3 OH, v: v=20/1.
In the present invention, the amide condensation reaction is carried out to obtain an amide condensation reaction liquid, and the present invention preferably carries out a post-treatment on the amide condensation reaction liquid to obtain a compound having a structure represented by formula 6. In the present invention, the post-treatment preferably includes: concentrating the amide condensation reaction liquid to obtain a residue; mixing and dissolving the residues and ethyl acetate, and then carrying out solid-liquid separation to obtain ethyl acetate filtrate; and washing, drying and removing the solvent from the ethyl acetate filtrate in sequence to obtain the compound with the structure shown in the formula 6. In the present invention, the concentration is preferably concentration under reduced pressure. The solid-liquid separation is preferably filtration, and the impurity is preferably removed by solid-liquid separation in the present invention. The washing is preferably: sequentially carrying out saturated NaHCO 3 aqueous solution washing, saturated NaCl aqueous solution washing and saturated KHSO 4 aqueous solution washing; each solution is preferably washed 3 times. In the present invention, the drying is preferably performed overnight with anhydrous sodium sulfate. In the present invention, the specific embodiment of the solvent removal is preferably concentration under reduced pressure.
After obtaining the compound of the structure represented by formula 6, the present invention mixes the compound of the structure represented by formula 6 with an organic solution of hydrogen chloride to perform an amino deprotection reaction (hereinafter referred to as a second amino deprotection reaction) to obtain the compound of the structure represented by formula 2.
In the present invention, in the second amino deprotection reaction, the organic solution of hydrogen chloride is preferably an ethyl acetate solution of hydrogen chloride. The molar concentration of hydrogen chloride in the organic solvent of hydrogen chloride is preferably 4mol/L. The ratio of the mass of the compound of the structure represented by formula 6 to the volume of the organic solvent of hydrogen chloride is preferably 1g:10mL.
In the present invention, the temperature of the second amino deprotection reaction is preferably room temperature, the time of the second amino deprotection reaction is preferably 4 hours, and the second amino deprotection reaction is preferably carried out under stirring. The second amino deprotection reaction is preferably detected by TLC using a developing reagent preferably CH 2Cl2/CH3 OH, v: v=20/1.
In the present invention, the second deprotection reaction of the second amino group is carried out to obtain a second deprotection reaction solution, and the present invention preferably carries out a post-treatment on the second deprotection reaction solution to obtain a compound having a structure represented by formula 2. In the present invention, the post-treatment preferably includes: the second deprotected reaction solution was first pumped out under reduced pressure with a water pump, and then washed 3 times with 20ml of anhydrous ethyl acetate. In the present invention, the coupling reagent is preferably O-benzotriazol-tetramethylurea Hexafluorophosphate (HBTU) at the time of the coupling reaction. The organic solvent is preferably N' N-Dimethylformamide (DMF). The molar ratio of Ma Qiansuan to the compound of the structure represented by formula 2 is preferably 1 (1.9 to 2.2), more preferably 1 (2 to 2.15). The mass ratio of Ma Qiansuan to the coupling agent is preferably 1:1.2. The invention has no special requirement on the dosage of the organic solvent, and ensures that the coupling reaction is carried out smoothly.
In the present invention, in the coupling reaction, the mixing is preferably: the Ma Qiansuan, the compound of the structure described by formula 2, the organic solvent and the coupling agent are mixed sequentially.
In the present invention, the temperature of the coupling reaction is preferably room temperature, and the time of the coupling reaction is preferably overnight.
In the invention, the coupling reaction is carried out to obtain a coupling reaction solution, and the invention preferably carries out post-treatment on the coupling reaction solution to obtain the compound with the structure shown in the formula 3. In the present invention, the post-treatment preferably includes: removing the solvent from the coupling reaction solution to obtain a solid residue; and separating the solid residues by column chromatography to obtain the compound with the structure shown in the formula 3. In the present invention, the specific embodiment of the solvent removal is preferably rotary evaporation. The column chromatographic separation is preferably carried out by adopting a reversed-phase C 18 column, the eluent is preferably a mixed solvent of water and acetonitrile, and the gradient elution is as follows: water: acetonitrile (v: v), wherein the volume content of acetonitrile is graded from 0% to 50%, the total eluting time is preferably 20min, the peak time is about 15min, and the eluent is removed after the eluent is obtained, wherein the specific implementation mode for removing the eluent is rotary evaporation.
After the compound with the structure shown in the formula 3 is obtained, the compound with the structure shown in the formula 3, the palladium-carbon catalyst and the organic solvent are mixed in hydrogen atmosphere to carry out reduction deprotection reaction, so that the derivative of the logenic acid with the structure shown in the formula 1 is obtained.
In the present invention, the organic solvent is preferably methanol in the reductive deprotection reaction. The mass ratio of the compound of the structure shown in formula 3 to the palladium-carbon catalyst is preferably 1:0.1. The invention has no special requirement on the dosage of the organic solvent, and ensures that the reduction deprotection reaction is carried out smoothly.
In the present invention, the temperature of the reductive deprotection reaction is preferably room temperature. The time for the reductive deprotection reaction is preferably overnight. The reductive deprotection reaction is preferably carried out under stirring.
In the invention, the reduction deprotection reaction is carried out to obtain a reduction deprotection reaction solution, and the invention preferably carries out post-treatment on the reduction deprotection reaction solution to obtain the derivative of the maleic acid with the structure shown in the formula 1. In the present invention, the post-treatment preferably includes: removing part of solvent from the reduction deprotection reaction liquid to obtain a wet solid residue; separating the wet solid residue by column chromatography to obtain the derivative of the maleic acid with the structure shown in the formula 1. In the present invention, the specific embodiment of the solvent removal is preferably rotary evaporation. The column chromatographic separation is preferably carried out by adopting a reversed-phase C 18 column, the eluent is preferably a mixed solvent of formic acid aqueous solution and acetonitrile, and the gradient elution is as follows: 0.5% by weight aqueous formic acid: acetonitrile (v: v), wherein the volume content of acetonitrile is gradient 0% -20%, the total elution time is 20min, the peak time is about 10min, and the eluent is removed after the eluent is obtained, and the specific implementation mode of removing the eluent is rotary evaporation.
The invention provides an application of the loganin acid derivative or the loganin acid derivative prepared by the preparation method of the technical scheme in preparing medicines for treating and/or preventing parkinsonism degenerative diseases or symptoms.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
The preparation of the derivative of maleic acid of the structure shown in formula 1 follows the scheme shown in figure 1:
3.05g (10 mmol) of a compound having a structure represented by formula 7 (Boc-Arg (NO 2)) was suspended in 100 to 120mL of anhydrous tetrahydrofuran to obtain a Boc-Arg (NO 2) suspension, 1.62g (12 mmol) of 1-hydroxybenzotriazole (HOBt) was added to the Boc-Arg (NO 2) solution under ice bath conditions, and then 2.27g (11 mmol) of Dicyclohexylcarbodiimide (DCC) was added thereto, followed by stirring for 20 to 30 minutes to obtain a first mixed solution.
2.42G (12 mmol) of the compound of formula 8 (Gly-OBzl. HCl) was added to the first mixed solution under ice bath to obtain a second mixed solution, 3-4 mL of N-methylmorpholine was added dropwise to adjust the pH of the second mixed solution to 9, the ice bath was removed, and the solution was stirred at room temperature overnight. The reaction was monitored by TLC (CH 2Cl2/CH3 OH, v: v=20/1), the reaction mixture obtained after the reaction was concentrated under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, and Dicyclohexylurea (DCU) was filtered off, and the impurities formed after the reaction was taken into the reaction by DCC after the reaction was completed. The filtrate was washed with saturated aqueous NaHCO 3 (12 mL. Times.3), saturated aqueous NaCl (12 mL. Times.3), saturated aqueous KHSO 4 (12 mL. Times.3), saturated aqueous NaCl (12 mL. Times.3), saturated aqueous NaHCO 3 (12 mL. Times.3), and saturated aqueous NaCl (12 mL. Times.3) in this order. The washed ethyl acetate phase was dried over anhydrous sodium sulfate overnight. Filtering, and concentrating the filtrate under reduced pressure to obtain 4g of white bubble substance, namely the compound (Boc-Arg (NO 2) -Gly-OBzl) with the structure shown in the formula 9.
2.5G of the compound of the structure shown in formula 9 (Boc-Arg (NO 2) -Gly-OBzl) is dissolved with 10-12 mL of methanol to obtain (Boc-Arg (NO 2) -Gly-OBzl) solution; then the pH value of the (Boc-Arg (NO 2) -Gly-OBzl) solution is adjusted to 13-14 by 2-3 mL of 2N NaOH aqueous solution under ice bath condition, stirring is carried out for 3.5-4 h, and TLC monitors that the reaction is completed (CH 2Cl2/CH3 OH, v: v=20/1). After the hydrolysis reaction is finished, the pH value of the mixture obtained by the reaction is adjusted to 7 by 3-4 mL of saturated KHSO 4 under the ice bath condition, and the methanol is removed by decompression concentration. The residue obtained by concentrating under reduced pressure was redissolved in pure water under ice bath, the pH was adjusted to 2 with 3mL of saturated KHSO 4, redissolved with ethyl acetate, extracted with ethyl acetate (12 mL. Times.3), and the combined ethyl acetate extract phases were dried over anhydrous sodium sulfate overnight. Filtering, concentrating the filtrate under reduced pressure to obtain about 2g of white solid, namely the compound (Boc-Arg (NO 2) -Gly) with the structure shown in the formula 4.
3.23G (10 mmol) of the compound of formula 10 (Boc-Asp (OBzl)) was dissolved in 100 to 120mL of anhydrous tetrahydrofuran to obtain Boc-Asp (OBzl) solution, 1.62g (12 mmol) of 1-hydroxybenzotriazole (HOBt) was added to the Boc-Asp (OBzl) solution under ice bath conditions, and then 2.27g (11 mmol) of Dicyclohexylcarbodiimide (DCC) was added thereto, followed by stirring for 20 to 30 minutes to obtain a third mixed solution;
4.6g (12 mmol) of the compound having the structure shown in formula 11 (Val-OBzl. Tos) was added to the third mixed solution under ice bath conditions to obtain a fourth mixed solution, 3-4 mL of N-methylmorpholine was added dropwise to adjust the pH of the fourth solution to 9, the ice bath was removed, and stirring was carried out at room temperature overnight. The reaction was monitored by TLC (CH 2Cl2/CH3 OH, v: v=20/1), after completion of the reaction was concentrated under reduced pressure, the residue was dissolved in 100mL ethyl acetate and DCU was filtered off. The filtrate was washed successively with saturated aqueous NaHCO 3 (12 mL. Times.3), saturated aqueous NaCl (12 mL. Times.3), saturated aqueous KHSO 4 (12 mL. Times.3), saturated aqueous NaCl (12 mL. Times.3), saturated aqueous NaHCO 3 (12 mL. Times.3), and saturated aqueous NaCl (12 mL. Times.3). The washed ethyl acetate phase was dried over anhydrous sodium sulfate overnight. Filtration and concentration of the filtrate under reduced pressure gave 4g of a yellow oil, which is the compound of formula 12 (Boc-Asp (OBzl) -Val-OBzl).
4G of the compound of formula 12 (Boc-Asp (OBzl) -Val-OBzl) was slowly dissolved in 40mL of an ethyl acetate solution of hydrogen chloride having a molar concentration of 4mol/L hydrogen chloride under ice-bath conditions. A solution of Boc-Asp (OBzl) -Val-OBzl was obtained, stirred for 4h, and TLC was monitored for reaction completion (CH 2Cl2/CH3 OH, v: v=20/1). The reaction solution obtained after the reaction is concentrated under reduced pressure by a water pump, the residue is redissolved by 20mL of anhydrous ethyl acetate and then concentrated under reduced pressure, and the mixture is repeated for 3 times to obtain yellow oily matter, namely the compound (Asp (OBzl) -Val-OBzl) with the structure shown in the formula 5.
10Mmol of a compound having a structure represented by formula 4 (Boc-Arg (NO 2) -Gly) was dissolved in 100 to 120mL of anhydrous tetrahydrofuran to obtain a Boc-Arg (NO 2) -Gly solution, 1.62g (12 mmol) of 1-hydroxybenzotriazole (HOBt) was added to the Boc-Arg (NO 2) -Gly solution under ice bath conditions, and then 2.27g (11 mmol) of Dicyclohexylcarbodiimide (DCC) was added thereto, followed by stirring for 20 to 30 minutes to obtain a fifth mixed solution.
12Mmol of the compound (Asp (OBzl) -Val-OBzl) with the structure shown in the formula 5 is added into the fifth mixed solution under ice bath to obtain a sixth mixed solution, 3-4 mL of N-methylmorpholine is added dropwise to adjust the pH value of the sixth mixed solution to 9, the ice bath is removed, and the mixture is stirred at room temperature overnight. The reaction was monitored by TLC (CH 2Cl2/CH3 OH, v: v=20/1), the reaction mixture obtained after the reaction was concentrated under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, and Dicyclohexylurea (DCU) was filtered off, and the impurities formed after the reaction was taken into the reaction by DCC after the reaction was completed. The filtrate was washed with saturated aqueous NaHCO 3 (12 mL. Times.3), saturated aqueous NaCl (12 mL. Times.3), saturated aqueous KHSO 4 (12 mL. Times.3), saturated aqueous NaCl (12 mL. Times.3), saturated aqueous NaHCO 3 (12 mL. Times.3), and saturated aqueous NaCl (12 mL. Times.3) in this order. The washed ethyl acetate phase was dried over anhydrous sodium sulfate overnight. Filtering, and concentrating the filtrate under reduced pressure to obtain a pale yellow solid, namely a compound (Boc-Arg (NO 2) -Gly-Asp (OBzl) -Val-OBzl) with a structure shown in a formula 6, wherein the yield is 55%. The mass spectrum of the compound with the structure shown in the formula 6 is shown in figure 2, and the nuclear magnetic hydrogen spectrum data is shown in figure 4.
4G of the compound of formula 6 (Boc-Arg (NO 2) -Gly-Asp (OBzl) -Val-OBzl) was slowly dissolved in 40mL of an ethyl acetate solution of hydrogen chloride having a molar concentration of hydrogen chloride of 4mol/L under ice-bath conditions. A solution of Boc-Arg (NO 2) -Gly-Asp (OBzl) -Val-OBzl was obtained, stirred for 4h and TLC monitored for reaction completion (CH 2Cl2/CH3 OH, v: v=20/1). The reaction solution obtained after the reaction is concentrated under reduced pressure by a water pump, the residue is redissolved by 20mL of anhydrous ethyl acetate and then concentrated under reduced pressure, and the mixture is repeatedly concentrated for 3 times to obtain white powder, namely the compound (Arg (NO 2) -Gly-Asp (OBzl) -Val-OBzl) with the structure shown in the formula 2, and the yield is 56.6%.
1G (2.6 mmol) of maleic acid and 1.21g (5.3 mmol) of a compound of the formula (Arg (NO 2) -Gly-Asp (OBzl) -Val-OBzl) were dissolved in 25 to 30mLN' N-Dimethylformamide (DMF) under ice bath conditions, 1.2g O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU) was added and reacted overnight at room temperature, the resulting reaction mixture was dried by spin-drying, and the residue was purified by a reversed phase C 18 column under the following conditions: reversed phase C 18 column, eluting with mixed solvent of water and acetonitrile, water: acetonitrile (v: v), wherein the volume content of acetonitrile is graded from 0% to 50%, the total elution time is 20min, the peak time is about 15min, and the flow rate of eluent is 50mL/min; spin-drying to obtain white powder, which is the compound (logenic acid-Arg (NO 2) -Gly-Asp (OBzl) -Val-OBzl) with the structure shown in formula 3, and the yield is 26%.
Adding a compound with a structure shown in formula 3 (maleic acid-Arg (NO 2) -Gly-Asp (OBzl) -Val-OBzl) and a palladium-carbon catalyst (the mass ratio of the compound with the structure shown in formula 3 to the palladium-carbon catalyst is 1:0.1) into 8-10 mL of methanol, stirring, introducing hydrogen overnight, filtering, and removing most of solvent, but not spin-drying, and preparing a column by using reversed-phase C 18 to obtain white powder, wherein the conditions are as follows: c 18, the eluent is: 0.5% by weight aqueous formic acid and acetonitrile, 0.5% by weight aqueous formic acid: acetonitrile (v: v), gradient elution: 0.5% by weight aqueous formic acid: acetonitrile (v: v), wherein the volume content of acetonitrile is gradient change of 0% -20%, total elution time is 20min, peak time is about 10min, and part of the acetonitrile is possibly salified, so that the logenic acid derivative with the structure shown in formula 1 is obtained, and the yield is 42%. The mass spectrum of the derivative of the logenic acid with the structure shown in the formula 1 is shown in figure 3, and the hydrogen nuclear magnetic spectrum is shown in figure 5. The ultraviolet spectra of the loganin acid and the loganin acid derivative of the structure shown in formula 1 are shown in fig. 6.
Test case
2. Examples of biological Activity
2.1 Experimental drugs
Test agent LA-1 (the derivative of logenic acid of the structure of formula 1 prepared in example 1)
2.2 Experimental reagents
Ceramide standard (860517), sphingomyelin standard (860584) (Avanti Polar Lipids, alabasma, USA); chloroform chromatography (11310278) (FISHER SCIENTIFIC, pittsburgh, PA, USA); 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP) (M0896), methanol chromatography (34860), pure ammonia (5.43830), ethanol (1012768) (Sigma, st.Louis, MO, USA); a ready-to-use immunohistochemical KIT (KIT-5001) (Maxim, fuzhou, china); TUNEL apoptosis kit (KGA 7061) (KeyGEN Biotech, nanJing, china); mouse dopamine (Mouse Dopamine) (DA) ELISA Kit (CSB-E08661 m) (Cusabio, houston, USA); mouse 3,4 dihydroxyphenylacetic acid (Mouse DOPAC)ELISA kit(MBS7269842)(Mybiosource,San Diego,USA);Homovanillic Acid(HVA)ELISA Kit(abx150352)(Abbexa,Cambridge,UK);5-HT ELISA Kit(ab285243)、Mouse IL-1beta antibody(ab197742)(Abcam,Cambridge,UK);Mouse TNF-alpha antibody(EM0183)(Finetest,Hubei,China); superoxide dismutase (SOD) assay kit (a 001-3) (Nanjing Jiancheng Bioengineering Institute, nanJing, china); glutathione peroxidase detection kit (BC 1195) (Solarbio, beijin, china); malondialdehyde (MDA) assay kit (S0131S) (Beyotime, shangHai, china); anti-PSD-95 Anti-body (3450) is available from CST company (CELL SIGNALING Technology, boston, USA); anti-TH Anti-ibody (ab 137869), anti-SYN Anti-ibody (ab 52636) are available from Abcam corporation (Abcam, cambridge, UK); the mouse anti-rabit IgG-FITC anti-body (sc-2359) was purchased from Santa Cruz, inc. (Santa Cruz, calif., USA).
Note that: the operations of the kit of the present invention are described with reference to the kit instructions.
2.3 Laboratory apparatus
FORMA 700 type ultralow temperature refrigerator, thermo company; YC-300L type medicine storage cabinet, department of middle Meiling low-temperature technology, ltd; direct-Q withpump ultrapure water instrument, millipore company; SWCJ-2FD type ultra-purification workbench: suzhou purification plant Co Ltd; 3K15 low temperature high speed centrifuge, sigma company; BS224 type electronic balance: beijing Sedris instruments systems Co., ltd; paraffin embedding machine, slicer, LEICA company, germany; berthold LB941 microplate type multifunctional microplate reader, berthold company; an Olympus inverted phase contrast microscope, olympus corporation, japan; zeiss LSM laser confocal microscope, zeiss company, germany; labmazeV3.0 animal behavior trace analysis system, beijing Zhongdi Ind technology development Co., ltd; ZS-KC open field box, beijing Zhongdi Ind technology development Co., ltd; the mouse pole-climbing experimental device is manufactured by Jiangsu Sianens biotechnology Co., ltd; 1260 high performance liquid chromatograph, agilent company, usa; 6410B triple quadrupole tandem mass spectrometer, agilent company, usa.
2.4 Laboratory animals
Female C57BL/6 mice, 8 weeks old; body weight 20±2g, supplied by the company, calves laboratory animal, inc., animal pass number: SCXK (su) 2021-0010, feeding in 22+ -2deg.C environment, and freely feeding and drinking water.
2.5 Experimental methods
2.5.1 Mouse model construction
After C57BL/6 mice are adaptively bred for one week, MPTP is injected into the abdominal cavity every day, the injection dosage is 30mg/kg,
The injection volume was 0.1mL/10g, and MPTP (Parkinson's disease modeling agent) was molded as Day0 for 5 consecutive days.
2.6 Experimental grouping
Normal group: c57BL/6 mice were fed normally, starting tail vein injection of equal volumes of normal saline at Day5, once daily, and continuously dosed to Ddy14 (n=6);
Model group (MPTP group): after the model of the PD model of the C57BL/6 mice was completed, an equal volume of physiological saline was injected into the tail vein at Day5, once daily, and administered continuously to Ddy14 (n=6);
Mptp+la-1 group: after the model of the PD of the C57BL/6 mouse is molded, the tail vein is started to be given to LA-1 (injection dose is 8.04 mg/kg) at Day5, the injection volume is 0.1mL once a Day, and the continuous administration is carried out until Ddy (N=6);
after the last administration for 12 hours, performing behavioural experiments such as open field experiments, pole climbing experiments and the like; after the behavioural experiment is finished, the mice are euthanized by a CO 2 method, the brain substantia nigra compact part of each group of mice is taken, and the TH content in the tissue samples of each group of mice is detected by an immunohistochemical experiment; TUNEL staining detects DA-capable neuronal apoptosis in each group of mouse tissues; ELISA for detecting DA, DOPAC, HVA, 5-HT, IL-1 beta and TNF-alpha content in mouse tissues; immunofluorescence experiment detection is carried out to observe the protein expression of SYN and PSD-95 in a substantia nigra region in a tissue section of a mouse; detecting the content of SOD, MDA, GSH-Px in the brain black matter region of the mouse by using the kit; LC-MS/MS experiments detect sphingomyelin and ceramide content in the substantia nigra region.
2.7 Open field experiments
ZS-KC open field is to observe and study the nerve and spirit change of experimental animals, and the size of the open field box of the mouse activity in the experimental is 500X 350mm after the experimental animals enter the open environment. The outer Zhou Ge is called along the wall lattice, and the rest lattices are central lattices. Each animal was placed in the center of the box bottom and all movement trajectories and performance parameters of the mice were collected using a camera and Labmaze V3.0.0 animal performance trajectory analysis system.
Before the experiment, each strain of mice is adapted to 1 week in a feeding room, during which the animals are caught and touched for about 5 minutes each day to adapt to the operation of an experimenter; mice were transferred into the test chamber for adaptation 30min before the start of the experiment to ensure that the mice activity tended to stabilize at the time of the experiment. The mice are placed back to the operators in the center of the open field, the camera is used for observing and recording the activities within 5 minutes, and a camera and Labmaze V3.0.0 animal behavior track analysis system are used for recording the tracks of the mice. After each animal experiment is finished, the faeces on the bottom plate of the open box are removed, 500mL/L ethanol is sprayed on the bottom of the open box and the bottom of the open box is wiped dry by clean gauze, so that the residual smell of the animal in the previous experiment is prevented from affecting the next experiment.
2.8 Pole climbing experiments
The tail part of the mouse is held, the head end of the mouse is downwards placed on the rod top, the four limbs of the mouse grasp the rod top, the total time from the beginning of the movement of the mouse from the rod top to the rod bottom to the whole grounding of the limbs of the mouse is recorded, and the recorded time is analyzed and compared to detect the action coordination of the mouse and record the gait abnormality of the climbing process. The pilot of this behavioural experiment was performed on mice before testing, each mouse was assayed three times, 5min apart, and the average was taken.
2.9 Immunohistochemistry
1) Baking slices: placing the prepared paraffin slice into an electrothermal constant temperature drying oven, and baking at 60 ℃ for 3 hours;
2) Performing conventional xylene dewaxing on the dried paraffin sections, hydrating with descending gradient ethanol, and washing with distilled water;
3) Antigen retrieval;
4) Dropwise adding 3wt% H 2O2, incubating for 10min at room temperature to inactivate endogenous enzymes, and washing with PBS for 3 times each for 3min;
5) Dripping normal goat immune serum into each slice for sealing, incubating for 10min at room temperature, removing excessive liquid, washing, dripping TH antibody (TH antibody is diluted in a ratio of 1:500), and refrigerating overnight at 4 ℃;
6) After being taken out from the refrigerator and restored to room temperature, the PBS is washed for 3 times, each time for 3min;
7) Dropwise adding a polymer reinforcing agent into each slice, washing with PBS for 3 times at room temperature for 3min each time;
8) Dripping enzyme-labeled anti-mouse-rabbit polymer into each slice, washing for 3 times at room temperature for 3min by PBS (phosphate buffer solution);
9) DAB color development, controlling reaction time under a microscope, counterstaining with hematoxylin, and washing with distilled water fully to stop color development;
10 Drying the slices by gradient alcohol dehydration, and sealing the slices by neutral resin, wherein the xylene is transparent;
11 Using a phase contrast microscope at 400 x magnification.
2.10 TUNEL staining
1) Dewaxing paraffin section by conventional method, and baking at 60deg.C for 60min. Xylene dewaxed 2 times, ethanol hydrated (100%, 95%, 80%, 75%); each time for 5min;
2) The sections were immersed in 1 XPBS and rinsed three times for 5min each;
3) Preparing a protease K working solution: the sample quantity is calculated and prepared in a concentrated way, and 10 mu L of 10 XProteinase K is added into 90 mu L of 1 XPBS for each sample, and the preparation is performed immediately;
4) mu.L of protease K working solution was added dropwise to each tissue section, and the reaction was carried out at 37℃for 30 minutes. The sections were immersed in 1 XPBS and rinsed three times for 5min each;
5) According to the types of samples, 100 mu L of DNase I reaction solution containing different activity units is prepared, and the preparation method is shown in Table 1:
Table 1 DNase I reaction solution preparation method
| Sample of | Paraffin section |
| Μ/100μL | 3000～5000Μ |
| DNase I (50 μm/. Mu.L) dosage | 60～100μL |
| DNase I Buffer usage | 40～0μL |
6) 100. Mu.L of DNase I (deoxyribonuclease I) reaction solution prepared according to Table 1 was added dropwise to one sample piece, treated at 37℃for 30 minutes,
7) Immersing the positive plate into 1X PBS for three times, and rinsing for 5min each time;
8) Preparing TdT enzyme reaction liquid: the sample numbers were calculated and formulated centrally (negative vs. photo not taken), each sample being: 1.0 μl of biotin-11-dM TP and 4.0 μl of TdT Enzyme were added at 45 μ L Equilibration Buffer, and the preparation was performed immediately, taking care of avoiding light;
9) The periphery of the sample is sucked by using a piece of absorbent paper, 50 mu L of TdT enzyme reaction solution is dripped on each sample, and the samples are placed into a temperature box to react for 60 minutes at 37 ℃ in a dark place. (Note: negative control sample was not added with TdT enzyme reaction solution)
10 Immersing the reacted sample piece in 1X PBS for three times, and rinsing for 5min each time, taking care of light shielding;
11 Mixing the strepitavidin-TRITC reagent with 45 mu L Labeling Buffer in an amount of 5 mu L per slice, and calculating the required total amount, namely, preparing the mixture immediately after use, taking care of avoiding light.
12 The periphery of the sample is sucked by a piece of absorbent paper, 50 mu L STREPTAVIDIN-TRITC marking liquid is dripped on each sample, and the samples are put into a wet box to react for 30min at 37 ℃ in a dark place.
13 Immersing the reacted sample piece in 1X PBS for three times, and rinsing for 5min each time, taking care of light shielding;
14 DAPI staining solution counterstained the nuclei and reacted at room temperature for 10min in the dark. DAPI dye was washed off and appropriate volume ratio caplets (glycerol: pbs=6:4) were added.
15 Fluorescence microscopy (630×): excitation wavelength 543nm and emission wavelength 571nm; (note: fluorescence is easily quenched, please observe the photograph as soon as possible).
2.11 Immunofluorescence
1) PBS washes the brain tissue sections of mice for 5min x 3 times;
2) Wiping off liquid around the tissue of the slice, and marking a circle at a position 2mm away from the tissue by utilizing the hydrophobic effect of an immunohistochemical pen so as to prevent dilution of various liquid components which are added dropwise;
3) Incubating and soaking 0.4% TritonX-100 at room temperature for 10min; PBS wash for 5min×3 times;
4) Antigen retrieval (1.5 min after the pressure limiting valve of the autoclave is inflated, and cold water is depressurized). Naturally cooling, and washing with PBS for 5min×3 times;
5) Sealing 5% sheep serum at 37deg.C for 10min;
6) Serum was discarded and primary antibody was added dropwise (SYN antibody diluted at 1:250; PSD-95 antibody diluted at 1:100 ratio), placed in a wet box, reacted overnight at 4 ℃;
7) The antibody reaction solution was blotted off and washed 5min 3 times with PBS;
8) Dripping a fluorescent-labeled secondary antibody (mouse anti-rabit IgG-FITC antibody is diluted in a ratio of 1:100), and incubating for 1-2 h in a wet box at 37 ℃ in a dark place;
9) The antibody reaction solution was blotted off and washed 5min 3 times with PBS;
10 90% of the glycerol sealing sheet is arranged in the light-shielding box;
11 Observation and photographing under 630 times of view of a laser confocal microscope.
2.12 ELISA (enzyme-Linked immunosorbent assay) for detecting DA (DA) content in tissue of black mass region of mouse
1) The tissues of the black area of the mice were washed with 1 XPBS, homogenized in 1 XPBS and stored overnight at-20 ℃. After membrane rupture by twice freeze thawing cycles, homogenizing is centrifuged for 5min at the temperature of between 2 and 8 ℃ and the concentration of 5000 Xg. The supernatant was immediately removed and examined.
2) The number of pre-coated strips required for one experiment was calculated and determined, the required strips were removed and placed in a 96 well frame, temporarily without strips, and the strips were returned to the aluminum foil bag for sealing and stored at 4 ℃.
3) Setting blank holes, standard substance holes and sample holes, respectively adding samples or standard substances with different concentrations into the corresponding holes according to 50 μl/hole, wherein the concentrations of the standard substances are as follows: 1000pg/mL, 250pg/mL, 80pg/mL, 20pg/mL, 5pg/mL, 0pg/mL.
4) Except for blank wells, 50 μl HRP-conjugate was added per well. 50. Mu.L of antibody was added to each well, mixed well and incubated at 37℃for 1h.
5) The liquid from each well was discarded and the wash repeated 3 times.
6) Each well was added with 50. Mu.L of each of the Substrate A and Substrate B, mixed well and incubated at 37℃for 15min.
7) Mu.L of stop solution was added to each well and the read data recorded immediately at 450 nm.
2.13 ELISA (enzyme-Linked immuno sorbent assay) for detecting DOPAC (DOPAC) content in tissues of black mass region of mice
1) Tissue homogenate: in this experiment, tissues were thoroughly cleared of excess blood by rinsing PBS in ice-cold. Tissue was minced and homogenized in 500 μl PBS using an ice-added glass homogenizer. The resulting suspension was subjected to ultrasound to further disrupt the cell membrane. Then, the homogenate was centrifuged at 1500 Xg for 15min, and the supernatant was taken for the experiment.
2) Setting a sample hole, a control hole and a standard hole, and adding 100 mu L of PBS, a sample or a standard into the sample hole, the control hole and the standard hole respectively; the standard concentration was 10ng/mL, 5ng/mL, 2.5ng/mL, 1.0ng/mL, 0.5ng/mL.
3) 50 Mu L of Conjugate is added to each well (except blank control well) and mixed well; sealing plates and incubating for 1h at 37 ℃;
4) Discarding the liquid in each hole, and washing the plate for 5 times;
5) 50. Mu.L of each of the Substrate A and Substrate B was added to each well; sealing plates and incubating for 20min at 37 ℃;
6) mu.L Stop Solution was added to each well and the read data recorded immediately at 450 nm.
2.14 ELISA (enzyme-Linked immunosorbent assay) for detecting HVA (high-level virus) content in tissue of black mass region of mouse
1) Tissue homogenate: the tissue was rinsed with ice-cold PBS and excess blood was removed. Tissue was minced and homogenized in PBS ice using a tissue homogenizer. The homogenate was centrifuged at 10000 Xg for 5min and the supernatant was collected.
2) Setting a sample hole, a control hole and a standard hole, and respectively adding 50 mu L of a sample, a standard dilution buffer or a standard into the sample hole, the control hole and the standard hole; the standard concentration was 200ng/mL, 66.7ng/mL, 22.2ng/mL, 7.41ng/mL, 2.47ng/mL.
3) 50 Mu L Detection Reagent A is added to each hole, and the mixture is incubated for 1h at 37 ℃;
4) Discarding the liquid in each hole, and washing for 3 times;
5) 100 mu L Detection Reagent B is added to each hole and incubated for 30min at 37 ℃;
6) Discarding the liquid in each hole, and washing for 5 times;
7) Adding 90 mu L TMB base solution into each hole, and incubating for 20min at 37 ℃;
8) mu.L of stop solution was added to each well and the read data recorded immediately at 450 nm.
2.15 ELISA (enzyme Linked immunosorbent assay) for detecting 5-HT content in tissue of black mass region of mouse
1) Tissue homogenate: the tissues were rinsed with ice-cold PBS to clear excess hemolysis. Appropriate amount of tissue was minced, homogenized in PBS, and ground with a glass homogenizer. To further destroy the tissue cells, the suspension is sonicated with an ultrasound cell disruptor. The homogenate was then centrifuged at 5000 Xg for 5min to obtain the supernatant.
2) The number of pre-coated strips required for one experiment was calculated and determined, the required strips were removed and placed in a 96 well frame, temporarily without strips, and the strips were returned to the aluminum foil bag for sealing and stored at 4 ℃.
3) Setting a standard hole, a sample hole and a control hole; samples or standards with different concentrations are respectively added into corresponding holes according to 50 mu L/hole, and the concentrations of the standards are as follows: 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 0ng/mL; 50 μl Biotin-detection antibody working solution was added immediately to each well, mixed well and incubated at 37deg.C for 45min.
4) The liquid from each well was discarded and washed 3 times.
5) Each well was added with 0.1mL SABC working solution, sealed and incubated at 37℃for 30min.
6) The liquid from each well was discarded and washed 5 times.
7) 50. Mu.L TMB substrate was added to each well, the plates were closed and incubated at 37℃for 30min in the absence of light.
8) Mu.L of Stop solution was added to each well and the read data recorded immediately at 450 nm.
2.16 ELISA (enzyme-Linked immunosorbent assay) for detecting IL-1 beta content in tissue of black mass region of mouse
1) Tissue homogenate: the tissue was first minced and thoroughly rinsed in PBS to remove blood in a ratio of 100mg wet tissue to 500. Mu.L of 1 Xfrozen cell extraction buffer, and the volume of extraction buffer added was adjusted accordingly. Incubate on ice for 20min. Centrifuging at 18000 Xg for 20min at 4 ℃; taking supernatant for detection.
2) The number of pre-coated strips required for one experiment was calculated and determined, the required strips were removed and placed in a 96 well frame, temporarily without strips, and the strips were returned to the aluminum foil bag for sealing and stored at 4 ℃.
3) Blank holes, standard substance holes and sample holes are arranged, and the concentration of the standard substance is as follows: 100pg/mL, 50pg/mL, 25pg/mL, 6.25pg/mL, 3.13pg/mL, 1.56pg/mL, 0pg/mL.
4) Samples or standards of different concentrations were added to the corresponding wells at 50. Mu.L/well, 50. Mu. L Antibody Cocktail were added to each well, the reaction wells were sealed with a sealing plate membrane and incubated at room temperature in a 400rpm rocker for 60min.
5) Each well plate was washed 3 times with 350. Mu.L of wash buffer, after each step the liquid was completely removed, after the last wash the plate was inverted and smeared on a clean paper towel to remove excess liquid.
6) 100 Mu L of a color reagent TMB solution was added to each well, the reaction well was sealed with a sealing plate film (white), and incubated at room temperature in a shaking plate at 400rpm for 10min in the absence of light. At low room temperature, a suitable extension of the incubation time is required, at which time incubation can be carried out until very significant colour changes occur in the standard and sample.
7) 100. Mu.L of stop solution was added to each well, and OD at 450nm was recorded.
2.17 ELISA (enzyme Linked immunosorbent assay) for detecting TNF-alpha content in tissue of black mass region of mouse
1) Tissue homogenate: the tissues of the brain black area of the mice were washed with pre-chilled PBS buffer to remove residual blood. Tissue was weighed and minced, homogenized on ice using a glass homogenizer. To further destroy tissue cells, the suspension may be sonicated with a sonicator. The homogenate was then centrifuged at 5000 Xg for 5min to give a supernatant.
2) Setting blank holes, standard substance holes and sample holes, and adding 100 mu L of sample and standard substance into each hole respectively; the standard substance concentration is: 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.625pg/mL, 7.812pg/mL, 3.906pg/mL, 0pg/mL.
3) Sealing plates, and incubating for 90min at 37 ℃;
4) Discarding the liquid in each hole, and washing the plate twice;
5) 100 mu L of Biotin-labeled antibodyworking solution is added into each hole, the plates are sealed, and the mixture is incubated for 90min at 37 ℃;
6) Discarding the liquid in each hole, and washing the plate for three times;
7) Adding 90 mu L of TMB Substrate into each hole, sealing the plates, and incubating for 20min at 37 ℃ in dark;
8) 50. Mu.L Stop Solution was added to each well and OD at 450nm was recorded.
2.18 Detection of SOD content in brain black matter region of mice by kit
1) Tissue homogenate: homogenizing the tissue by using PBS, wherein the weight of the tissue accounts for 10% of the weight of the homogenate or lysate; after homogenization or lysis, the supernatant was centrifuged at 2500rpm for 10min and used for subsequent determination.
2) Adding reagents according to Table 2
TABLE 2 types and amounts of reagents
2.19 Detection of MDA content in brain black matter region of mice by kit
1) Tissue homogenate: to homogenize the tissue using PBS, the amounts and types of reagents in tables 4 and 5, the weight of tissue was 10% of the homogenate or lysate; after homogenization or lysis, 10000g was centrifuged for 10min and the supernatant was taken for subsequent determination.
2) Adding 0.1mL of homogenate and PBS solution into a centrifuge tube or other suitable containers to serve as blank control, adding 0.1mL of standard substances with different concentrations to be used for making a standard curve, and adding 0.1mL of sample to be used for determination; then 0.2mL of MDA detection working solution was added. The standard substance concentration is: 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 50. Mu.M; table 3 sets up the detection reaction system:
TABLE 3 detection reaction System
| Project | Blank control | Standard substance | Sample of |
| PBS | 0.1mL | - | - |
| Standard substance | - | - | |
| Sample to be measured | - | - | 0.1mL |
| MDA reaction working solution | 0.2mL | 0.2mL | 0.2mL |
3) After mixing, the mixture was heated in a boiling water bath for 15min. If the heating block is used for heating, the centrifugal tube cover is tightly pressed by a weight;
4) Cooled to room temperature in a water bath, and centrifuged at 1000g for 10min at room temperature. 200. Mu.L of the supernatant was added to a 96-well plate, and absorbance was measured at 532nm using a microplate reader.
2.20 Detection of GSH-Px content in brain black matter region of mice by kit
1) Preheating for 30min by an enzyme-labeled instrument, adjusting the wavelength to 412nm, and zeroing by distilled water;
2) Diluting 80. Mu. Mol/mL standard to 0.08. Mu. Mol/mL;
3) Reagents were added as in table 4.
TABLE 4 types and amounts of reagents
Fully and uniformly mixing, centrifuging at 4000rpm at normal temperature for 5min, and taking supernatant.
TABLE 5 types and amounts of reagents
| Dilution liquid | - | - | - | 100 |
| Supernatant fluid | 100 | 100 | - | - |
| Standard solution | - | - | 100 | - |
| Reagent IV | 100 | 100 | 100 | 100 |
| Reagent five | 25 | 25 | 25 | 25 |
Mixing well, standing at room temperature for 15min, and measuring absorbance at 412 nm.
2.21 LC-MS/MS
Standard solution preparation A stock solution of 1. Mu. Mo1/mL was prepared by dissolving the ceramide and sphingomyelin standard in chloroform/methanol (2/1, v/v) solvents, respectively. Stock solutions were diluted with mobile phase A/B (1/1, v/v) to standard use solutions of different concentrations as follows: ceramide: 100pmol/mL, 200pmol/mL, 400pmol/mL, 800pmol/mL, 1600pmol/mL, 3200pmol/mL; sphingomyelin: 1nmo1/mL, 2nmo1/mL, 4nmo1/mL, 8nmo1/mL, 16nmo1/mL, 32nmo1/mL; in addition, an equivalent amount of ceramide internal standard was added, and the internal standard concentration was 125pmol/mL, respectively.
Chromatographic conditions
Liquid chromatographic column: agilent RX-SIL (2.1 mm. Times.100 mm,1.8 μm); mobile phase a: chloroform/methanol/ammonia (89.5/10/0.5, v/v); mobile phase B: chloroform/methanol/water/ammonia (55/39/5/0.5, v/v); gradient elution, elution time and ratio are shown in the following table: flow rate: 0.3mL/min; the column temperature is kept at 25 ℃; the sample injection amount was 5. Mu.L. Gradient elution procedure is as in table 6:
TABLE 6 liquid chromatography column elution procedure
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 3 | 95 | 5 |
| 27 | 70 | 30 |
| 32 | 70 | 30 |
| 37 | 30 | 70 |
| 44 | 30 | 70 |
Mass spectrometry conditions
The Agilent triple quadrupole mass spectrometer adopts a parent ion scanning mode to screen parent ions, and adopts an MRM mode for quantification. Operating parameters: drying gas flow rate: 9L/min; drying gas temperature: 300 ℃; atomization gas pressure: 40psi.
2.22 Data analysis
Data were analyzed and plotted using GRAPHPAD PRISM (Version 5.01), and collated using Adobe Illustrator CS6 (Version 16.0.0). All data are expressed in means±sd, and statistical differences between groups were examined using one-way ANOVA and Tukey's, with P values less than 0.05 considered significant differences.
3. Experimental results
3.1 As shown in FIG. 7, FIG. 7 shows the effect of LA-1 on PD mouse behavior, specifically: c57BL6 mice were injected with MPTP (30 mg/kg intravenous injection) for 5 days, followed by LA-1 (8.04 mg/kg intravenous injection) for 10 days. A in fig. 8 is total distance statistics, B in fig. 7 is center crossing count statistics, C in fig. 7 is center distance statistics, and D in fig. 7 is feeding statistics; the detection was performed using an open field test. Bar test (E). Results are expressed as mean ± standard deviation (n=6). * p <0.05, p <0.01.
In the open field experiment, compared with the Control group, the total moving distance, the number of times of crossing the center, the center moving distance and the number of erection times of the MPTP group mice are obviously reduced; compared with the MPTP group, the total moving distance, the center crossing times, the center moving distance and the erection times of the MPTP+LA-1 group mice are obviously increased; in the pole-climbing experiment, compared with the Control group, the pole-climbing time of the MPTP group mice is obviously increased, and compared with the MPTP group, the pole-climbing time of the MPTP+LA-1 group mice is obviously reduced.
3.2 As shown in FIG. 8, FIG. 8 shows the effect of LA-1 on SN tissue TH expression in PD mice. The method comprises the following steps: c57BL6 mice were injected with MPTP (30 mg/kg intravenous injection) for 5 days, followed by LA-1 (8.04 mg/kg intravenous injection) for 10 days. Immunohistochemical assay SN (bar=50 μm) mice TH, test results are shown as a and B in fig. 8. Results are expressed as mean ± standard deviation (n=6). * p <0.05, p <0.01.
In the immunohistochemical experiment, compared with the Control group, the average optical density of the TH positive region of the substantia nigra compact part of the MPTP group mice is obviously reduced; compared with the MPTP group, the average optical density of the TH positive region of the substantia nigra compact part of the mice in the MPTP+LA-1 group is obviously increased.
3.3 As shown in FIG. 9, FIG. 9 shows the effect of LA-1 on SN expression in PD mice. The method comprises the following steps: c57BL6 mice were injected with MPTP (30 mg/kg intravenous injection) for 5 days, followed by LA-1 (8.04 mg/kg intravenous injection) for 10 days. The percentage of TUNEL positive cells was calculated by TUNEL staining (bar=50 μm) as the result of a in fig. 9, and as the result of B in fig. 9. Results are expressed as mean ± SD (n=6). * p <0.05, p <0.01.
In TUNEL experiments, compared with the Control group, the number of the black matrix TUNEL positive cells of the MPTP group mice is obviously increased; compared with the MPTP group, the number of the black matrix TUNEL positive cells of the MPTP+LA-1 group mice is obviously reduced. These results indicate that the compound LA-1 can inhibit apoptosis and play a certain role in protection.
3.4 As shown in FIG. 10, FIG. 10 shows the effect of LA-1 on the expression of SYN in SN tissue of PD mice. The method comprises the following steps: c57BL6 mice were given MPTP (30 mg/kg i.p.) for 5 days, followed by LA-1 (8.04 mg/kg intravenous drip) for 10 consecutive days. SYN expression in mouse SN was detected with confocal microscopy (a in fig. 10) (bar=50 μm), average fluorescence intensity (B in fig. 10), expressed as mean ± SD (n=6). * p <0.05, p <0.01.
In the immunofluorescence experiment, compared with the Control group, the average fluorescence intensity of the black matrix SYN positive region of the MPTP group mice is obviously increased; compared with the MPTP group, the average fluorescence intensity of the black matrix SYN positive region of the MPTP+LA-1 group mice is obviously reduced.
3.5 As shown in FIG. 11, FIG. 11 shows the effect of LA-1 on the expression of PSD-95 in SN tissue of PD mice. The method comprises the following steps: c57BL6 mice were given MPTP (30 mg/kg i.p.) for 5 days, followed by LA-1 (8.04 mg/kg intravenous drip) for 10 consecutive days. Confocal microscopy (a in fig. 11) (bar=50 μm) detected expression of PSD-95 in mouse SN, average fluorescence intensity (B in fig. 11). Results are expressed as mean ± standard deviation (n=6). * p <0.05, p <0.01.
In the immunofluorescence experiment, compared with the Control group, the average fluorescence intensity of the black matter PSD-95 positive region of the MPTP group mice is obviously increased; compared with the MPTP group, the average fluorescence intensity of the black matter PSD-95 positive region of the MPTP+LA-1 group mice is obviously reduced.
3.6 As shown in FIG. 12, FIG. 12 shows the effect of LA-1 on SN tissue DA, DOPAC, HVA, 5-HT, IL-1β, TNF- α expression in PD mice, specifically: c57BL6 mice were injected with MPTP (30 mg/kg intravenous injection) for 5 days, followed by LA-1 (8.04 mg/kg intravenous injection) for 10 days. ELISA method detected SN mice DA (A in FIG. 12), DOPAC (B in FIG. 12), HVA (C in FIG. 12), 5-HT (D in FIG. 12), IL-1β (E in FIG. 13), TNF- α (F in FIG. 13). Results are expressed as mean ± standard deviation (n=6). * p <0.05, p <0.01.
Compared with the Control group, the black matrix DA, DOPAC, HVA and 5-HT content of the MPTP group mice are obviously reduced, and the IL-1 beta and TNF-alpha content are obviously increased; compared with the MPTP group, the black matrix DA, DOPAC, HVA and 5-HT content of the MPTP+LA-1 group mice are obviously increased, and the IL-1 beta and TNF-alpha content are obviously reduced.
3.7 As shown in FIG. 13, FIG. 13 shows the effect of LA-1 on SOD, MDA, GSH-px expression in SN tissue of PD mice. The method comprises the following steps: c57BL6 mice were injected with MPTP (30 mg/kg intravenous injection) for 5 days, followed by LA-1 (8.04 mg/kg intravenous injection) for 10 days. SOD (A in FIG. 13), MDA (B in FIG. 13), GSH-px (C in FIG. 13). Results are expressed as mean ± standard deviation (n=6). * p <0.05, p <0.01.
Compared with the Control group, the activity of the black matrix SOD and GSH-px of the MPTP group (model group) mice is obviously reduced, and the MDA content is obviously increased; compared with the MPTP group, the activity of the black matrix SOD and GSH-px of the MPTP+LA-1 group mice is obviously increased, and the MDA content is obviously reduced.
3.8 As shown in FIG. 14, FIG. 14 shows the effect of LA-1 on SN tissue sphingomyelin and ceramide in PD mice. The method comprises the following steps: c57BL6 mice were injected with MPTP (30 mg/kg intravenous injection) for 5 days, followed by LA-1 (8.04 mg/kg intravenous injection) for 10 days. LC-MS detected sphingomyelin (A in FIG. 14) and ceramide (B in FIG. 14) in mouse SN. Results are expressed as mean ± standard deviation (n=6). * p <0.05, p <0.01.
Compared with the Control group, the content of the black sphingomyelin and the ceramide of the MPTP group mice is obviously increased; compared with the MPTP group, the content of the black sphingomyelin and the ceramide in the mice in the MPTP+LA-1 group is obviously reduced.
Literature "Loganin substantially ameliorates molecular deficits,pathologies and cognitive impairment in amouse model ofAlzheimer's disease.Aging(AlbanyNY)"(Nie L,He K,Xie F,Xiao S,Li S,Xu J,Zhang K,Yang C,Zhou L,Liu J,Zou L,Yang X..2021Oct23;13(20):23739-23756.) discloses an activity evaluation of loganin in 3xTg-AD (triple transgenic) mice, wherein loganin is used at a dose of 20mg/kg; in the test examples of the present invention, APP/PS1 AD mice were used, and the amount of the derivative of loganin having the structure of formula 1 prepared in example 1 was 8.04mg/kg, which is a 2.49-fold decrease in the amount of the derivative of loganin having the structure of formula 1 prepared in example 1, compared to the above-mentioned document. The APP/PS1 AD mice used in the test examples of the present invention were double-transgenic 2xTg, which is different from the triple-transgenic 3xTg reported in the above-mentioned document in that the mutated genes are different, but the model mice in the above-mentioned document and the model mice in the present invention are all characterized in that the mutated APP genes, which all contain AD characteristics, cause amyloid plaques to appear in the brain (see "comparative study of APP/PS1 double-transgenic and APP/PS1/Tau triple-transgenic alzheimer disease model mice [ D ]. Zhou Yi, university of south-middle, 2012"). Thus, the present invention employs an effective dose of the loganin derivative for the treatment of model mice, which is reduced by a factor of 2.49 in terms of the dose used, compared to loganin employed in the above-mentioned documents.
The activity result shows that the loganin acid derivative with the structure shown in the formula 1 can protect or repair the damage of the nervous system, and has good treatment effect prospect on the degenerative diseases or symptoms of the parkinsonism.
Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be obtained according to the present embodiments without departing from the scope of the invention.
Claims (10)
1. A derivative of maleic acid, characterized by having a structure represented by formula 1:
2. a process for the preparation of a derivative of logenic acid as defined in claim 1, comprising the steps of:
Mixing loganin acid, a compound with a structure shown in a formula 2, a coupling reagent and an organic solvent for coupling reaction to obtain a compound with a structure shown in a formula 3;
And in a hydrogen atmosphere, mixing the compound with the structure shown in the formula 3, a palladium-carbon catalyst and an organic solvent for reduction and deprotection reaction to obtain the derivative of the maleic acid with the structure shown in the formula 1.
3. The preparation method according to claim 2, wherein the preparation method of the compound of the structure represented by formula 2 comprises the steps of:
Mixing a compound with a structure shown in a formula 4, a compound with a structure shown in a formula 5, a condensation reagent, a dehydrating agent, an organic base reagent and an organic solvent for carrying out an amide condensation reaction to obtain a compound with a structure shown in a formula 6;
And mixing the compound with the structure shown in the formula 6 with an organic solution of hydrogen chloride to perform amino deprotection reaction to obtain the compound with the structure shown in the formula 2.
4. A method according to claim 3, wherein the method for producing the compound of the structure represented by formula 4 comprises the steps of:
mixing a compound with a structure shown in a formula 7, a compound with a structure shown in a formula 8, a condensation reagent, a dehydrating agent, an organic alkali reagent and an organic solvent for condensation reaction to obtain a compound with a structure shown in a formula 9;
and mixing the compound with the structure shown in the formula 9, an organic solvent and an inorganic alkali solution for hydrolysis reaction to obtain the compound with the structure shown in the formula 4.
5. A method according to claim 3, wherein the method for producing the compound of the structure represented by formula 5 comprises the steps of:
mixing a compound with a structure shown in a formula 10, a compound with a structure shown in a formula 11, a condensation reagent, a dehydrating agent, an organic alkali reagent and an organic solvent for condensation reaction to obtain a compound with a structure shown in a formula 12;
And mixing the compound with the structure shown in the formula 12 with an organic solution of hydrogen chloride to perform amino deprotection reaction to obtain the compound with the structure shown in the formula 5.
6. The preparation method according to claim 2, wherein the molar ratio of Ma Qiansuan to the compound of the structure represented by formula 2 is 1 (1.9-2.2).
7. The process according to claim 3, wherein the molar ratio of the compound of the structure represented by formula 4 to the compound of the structure represented by formula 5 is 1 (1.1 to 1.3).
8. The preparation method according to claim 4, wherein the mass ratio of the compound having the structure represented by formula 7 to the compound having the structure represented by formula 8 is 1 (1.1 to 1.3).
9. The process according to claim 5, wherein the molar ratio of the compound of the structure represented by formula 10 to the compound of the structure represented by formula 11 is 1 (1.1 to 1.3).
10. Use of a derivative of loganin as claimed in claim 1 or prepared by a method as claimed in any of claims 2 to 9 in the manufacture of a medicament for the treatment and/or prophylaxis of degenerative diseases or conditions of the parkinsonism.
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