CN115651062A - Strychnos acid derivative and preparation method and application thereof - Google Patents
Strychnos acid derivative and preparation method and application thereof Download PDFInfo
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- CN115651062A CN115651062A CN202211264173.5A CN202211264173A CN115651062A CN 115651062 A CN115651062 A CN 115651062A CN 202211264173 A CN202211264173 A CN 202211264173A CN 115651062 A CN115651062 A CN 115651062A
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Abstract
The invention belongs to the technical field of medicineParticularly relates to a loganin acid derivative and a preparation method and application thereof. The invention provides a loganin acid derivative which has a structure shown in a formula 1. The animal model experiment shows that: after a loganin acid derivative with the structure shown in formula 1 provided by the invention is adopted to treat a model mouse: in an immunohistochemical experiment, the average optical density of the positive region of TH of the substantia nigra compact part of a treated mouse is obviously increased; the activity of the treated mouse substantia nigra SOD and GSH-px is obviously improved, and the content of MDA is obviously reduced; the content of the sphingomyelin and ceramide in the treated mice is obviously reduced. The 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 Parkinson nervous system degenerative disease or disease.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a loganic acid derivative, and a preparation method and application thereof.
Background
Neurodegenerative diseases are characterized pathologically by progressive, irreversible dysfunction and neuronal loss, behaviorally by a decline or even loss of learning and memory, and are the main features of diseases such as Alzheimer's Disease (AD) and Parkinson's Disease (PD). The Parkinson is one of common clinical diseases, seriously influences the health of middle-aged and elderly people, and is mainly clinically manifested by resting tremor, bradykinesia, muscular rigidity, gait disturbance of postures and the like. Parkinson's disease is a neurodegenerative disease second only to Alzheimer's disease, and the cause is unclear.
At present, the treatment of the Parkinson's disease becomes a key direction of research in the medical field, and the treatment medicines mainly comprise western medicines, wherein a levodopa preparation is one of the most effective medicines, but the side effect is obvious, and the side effect is larger when the dosage is larger. Therefore, there is an urgent need to develop effective drugs for treating parkinson.
Disclosure of Invention
The invention aims to provide a maleic acid derivative, a preparation method and an application thereof.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a loganin acid derivative which has a structure shown in a formula 1:
the invention provides a preparation method of the loganin acid derivative in the technical scheme, which comprises the following steps:
mixing loganic 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;
in a hydrogen atmosphere, mixing the compound with the structure shown in the formula 3, a palladium-carbon catalyst and an organic solvent for carrying out reduction deprotection reaction to obtain the loganic acid derivative with the structure shown in the formula 1.
Preferably, the preparation method of the compound having the structure shown in 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 to perform 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 and an organic solution of hydrogen chloride for carrying out amino deprotection reaction to obtain the compound with the structure shown in the formula 2.
Preferably, the preparation method of the compound having the structure represented by 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 base 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 having the structure shown in 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 base 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 and an organic solution of hydrogen chloride for carrying out amino deprotection reaction to obtain the compound with the structure shown in the formula 5.
Preferably, the molar ratio of the loganic acid to the compound with the structure shown in the formula 2 is 1 (1.9-2.2).
Preferably, the molar ratio of the compound having the structure represented by formula 4 to the compound having the structure represented by formula 5 is 1 (1.1 to 1.3).
Preferably, 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).
Preferably, the molar ratio of the compound having the structure represented by formula 10 to the compound having the structure represented by formula 11 is 1 (1.1 to 1.3).
The invention provides application of the loganin acid derivative in the technical scheme or the loganin acid derivative prepared by the preparation method in the technical scheme in preparation of a medicine for treating and/or preventing the Parkinson nervous system degenerative disease or disorder.
The invention provides a loganin acid derivative which has a structure shown in a formula 1. The loganin acid derivative provided by the invention couples loganin acid and a short peptide, wherein the short peptide is a peptide sequence with an arginine-glycine-aspartic acid (RGD) motif, and the short peptide is used for integrin (integrin) and especially alpha V β 3 Integrins exhibit strong affinity. Integrins are transmembrane glycoproteins involved in cell interactions 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 loganin acid derivative obtained by coupling loganin acid and short peptide is coupled by RGD sequence peptide, and has targeting effect. The animal model experiment shows that: after a loganin acid derivative with the structure shown in formula 1 provided by the invention is adopted to treat a model mouse: in an open field experiment, the total moving distance, the center crossing times, the center moving distance and the erection times of the treated mice are obviously increased; in the pole climbing experiment, the pole climbing time of the treated mice is obviously reduced; in an immunohistochemical experiment, the average optical density of a TH positive area of a substantia nigra compact part of a treated mouse is obviously increased; in the TUNEL experiment, the number of TUNEL positive cells in the substantia nigra of the treated mice is obviously reduced; in an immune fluorescence experiment, the average fluorescence intensity of a treated mouse substantia nigra SYN positive area is obviously reduced, and the average fluorescence intensity of a treated mouse substantia nigra PSD-95 positive area is obviously reduced; the contents of DA, DOPAC, HVA and 5-HT of the substantia nigra of the treated mice are obviously increased, and the contents of IL-1beta and TNF-alpha are obviously reduced; the activity of the treated mouse substantia nigra SOD and GSH-px is obviously increased, and the MDA contentThe obvious reduction is achieved; the content of the sphingomyelin and ceramide in the treated mice is obviously reduced. The activity results show 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 Parkinson nervous system degenerative diseases or symptoms.
The invention provides a preparation method of the loganic acid derivative in the technical scheme, which comprises the following steps: mixing the loganic acid, the compound with the structure shown in the formula 2, a coupling reagent and an organic solvent for coupling reaction to obtain a compound with the structure shown in the formula 3; 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 deprotection reaction to obtain the loganic acid derivative with the structure shown in the formula 1. According to the preparation method provided by the invention, the loganin acid and the full-protection tetrapeptide are coupled and then hydrogenated to successfully obtain the tetrapeptide modified loganin acid consisting of arginine-glycine-aspartic acid-valine, so that the damage of a nervous system can be effectively protected or repaired, and the preparation method has a good treatment effect on the Parkinson nervous system degenerative diseases or diseases. And the preparation method is simple and 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 formula 6 prepared according to an example of the present invention;
FIG. 3 is a mass spectrum of a loganic acid derivative prepared according to an embodiment of the present invention;
FIG. 4 is a hydrogen nuclear magnetic spectrum of a compound of formula 6 prepared according to an example of the present invention;
FIG. 5 is a graph of the hydrogen nuclear magnetic spectrum of a loganic acid derivative prepared according to an embodiment of the present invention;
FIG. 6 is a UV spectrum of a loganic acid derivative prepared according to an example of the present invention;
FIG. 7 shows the effect of the loganin acid derivatives prepared in the present example on the behavioural characteristics of PD mice;
FIG. 8 is a graph showing the effect of a loganin acid derivative prepared according to an embodiment of the present invention on TH expression in SN tissue of PD mice;
FIG. 9 is a graph showing the results of a loganic acid derivative prepared according to an embodiment of the present invention on SN in PD mice;
FIG. 10 is a graph showing the effect of a loganin acid derivative prepared according to an embodiment of the present invention on the expression of SYN in SN tissue of PD mice;
FIG. 11 is a graph showing the effect of a loganin acid derivative prepared according to an embodiment of the present invention on the expression of PSD-95 in SN tissue of PD mice;
FIG. 12 is a graph showing the effect of a loganin acid derivative prepared according to an embodiment of the present invention on the expression of SN tissues DA, DOPAC, HVA, 5-HT, IL-1 β, TNF- α in PD mice;
FIG. 13 shows the effect of the marchanoic acid derivatives prepared according to the example of the present invention on the expression of SOD, MDA, GSH-px in SN tissue of PD mice;
FIG. 14 shows the effect of a loganin acid derivative prepared according to the present invention on SN tissue sphingomyelin and ceramide in PD mice.
Detailed Description
The invention provides a loganin acid derivative which has a structure shown in a formula 1:
the invention provides a preparation method of the loganic acid derivative in the technical scheme, which comprises the following steps:
mixing the loganic acid, the compound with the structure shown in the formula 2, a coupling reagent and an organic solvent for coupling reaction to obtain a compound with the structure shown in the formula 3;
in a hydrogen atmosphere, mixing the compound with the structure shown in the formula 3, a palladium-carbon catalyst and an organic solvent for carrying out reduction deprotection reaction to obtain the loganic acid derivative with the structure shown in the formula 1.
In the present invention, all the preparation starting materials/components are commercially available products well known to those skilled in the art, unless otherwise specified.
According to the invention, the loganic acid, the compound with the structure shown in the formula 2, the coupling reagent and the organic solvent are mixed for coupling reaction, so that the compound with the structure shown in the formula 3 is obtained.
In the present invention, the formula of the loganic acid is shown in formula 13:
in the present invention, the method for preparing the compound having 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 to perform 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 and an organic solution of hydrogen chloride for carrying out amino deprotection reaction to obtain the compound with the structure shown in the formula 2.
The compound with the structure shown in the formula 4, the compound with the structure shown in the formula 5, a condensation reagent, a dehydrating agent, an organic base reagent and an organic solvent are mixed for carrying out an amide condensation reaction, so as to obtain the compound with the structure shown in the formula 6.
In the present invention, the method for preparing the compound having 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 base 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.
The compound of formula 7, the compound of formula 8, a condensation reagent, a dehydrating agent, an organic base reagent and an organic solvent are mixed and subjected to a condensation reaction (hereinafter referred to as a first condensation reaction) to obtain a compound of formula 9.
In the present invention, the first condensation reaction: 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: the molar ratio of the compound having a structure represented by formula 7 to the compound having a structure represented by formula 8 is preferably 1 (1.1 to 1.3), more preferably 1. The molar ratio of the compound having the structure represented by formula 7 to the condensing agent is preferably 1.2. The molar ratio of the compound having a structure represented by formula 7 to the dehydrating agent is preferably 1.1. The amount of the organic base reagent is preferably adjusted to a pH value of 8 to 9 in a mixed solution obtained by mixing a compound having a structure represented by formula 7, a compound having a structure represented by formula 8, a condensation reagent, a dehydrating agent and an organic solvent. The invention has no special requirement on the dosage of the organic solvent, and the first condensation reaction is ensured to be smoothly carried out.
In the present invention, the first condensation reaction: 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 a formula 7; under the condition of ice-water bath, 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 to obtain a first mixed solution; mixing the first mixed solution and a compound with the structure shown in the formula 8 to obtain a second mixed solution; and dropwise adding the organic alkali reagent into the second mixed solution, adjusting the pH value of the second mixed solution to 8-9, and withdrawing the mixed solution from the ice water bath.
In the inventionThe temperature of the first condensation reaction is preferably room temperature. The time for the first condensation reaction is preferably overnight. The first condensation reaction is carried out under stirring. According to the invention, the first condensation reaction is preferably detected by TLC, and the reagent used by TLC is preferably CH 2 Cl 2 /CH 3 OH,v:v＝20/1。
In the present invention, the first condensation reaction is carried out to obtain a first condensation reaction liquid, and in the present invention, it is preferable to carry out a post-treatment of the first condensation reaction liquid 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 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 in the invention, impurity impurities generated after DCC participates in the reaction are preferably removed through solid-liquid separation. The washing is preferably: saturated NaHCO was performed sequentially 3 Washing with water solution, washing with saturated NaCl solution, and saturating with KHSO 4 Washing with an aqueous solution; each solution was washed 3 times. In the present invention, the drying is preferably performed for 2 hours or more using anhydrous sodium sulfate. In the present invention, a 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, and the compound with the structure shown in the formula 4 is obtained.
In the present invention, the organic solvent is preferably methanol in the hydrolysis reaction. The organic base solution is preferably an aqueous NaOH solution. The molar concentration of the inorganic base solution is preferably 2mol/L. The invention has no special requirement on the dosage of the organic solvent, and the compound with the structure shown in the formula 9 can be completely dissolved. The inorganic alkali solution is preferably used in an amount such that the pH of an organic solution of the compound having the structure represented by formula 9 is adjusted to 13 to 14.
In the present invention, the mixing is preferably performed by: 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 (2) mixing the inorganic alkali solution with 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 performed under stirring in an ice-water bath. In the present invention, the time for the hydrolysis reaction is preferably 3.5 to 4 hours. The invention preferably uses TLC to detect the hydrolysis reaction, and the developing agent used by TLC is preferably CH 2 Cl 2 /CH 3 OH,v:v＝20/1。
In the present invention, the hydrolysis reaction is performed to obtain a hydrolysis reaction solution, and in the present invention, the hydrolysis reaction solution is preferably subjected to a post-treatment to obtain a compound having a structure represented by formula 4. In the present invention, the post-treatment preferably comprises: under the condition of ice-water bath, the hydrolysis reaction solution and saturated KHSO are mixed 4 Mixing and adjusting the pH value of the reaction solution to 7; removing the organic solvent from the hydrolysis reaction with the pH value of 7 to obtain a residue; redissolving the residue in pure water in an ice-water bath, and adding saturated KHSO 4 Adjusting the pH value of the residue to 2 by using the solution, and re-dissolving the residue by using an ethyl acetate solution; extracting the residue with the pH value of 2 to obtain an extracted organic phase; and washing, drying and removing the extraction solvent from the extracted organic phase in sequence to obtain the compound with the structure shown in the formula 4. The specific embodiment of removing the organic solvent is preferably concentration under reduced pressure. The extractant for extraction is preferably ethyl acetate. The number of extractions is preferably 3, and the organic phases are combined and dried. The drying is preferably carried out overnight using anhydrous sodium sulfate. The specific embodiment of the extraction solvent removal is preferably concentration under reduced pressure.
In the present invention, the method for preparing the compound having 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 base 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 and an organic solution of hydrogen chloride for carrying out amino deprotection reaction to obtain the compound with the structure shown in the formula 5.
The compound of formula 10, the compound of formula 11, a condensation reagent, a dehydrating agent, an organic base reagent and an organic solvent are mixed to carry out a condensation reaction (second condensation reaction) to obtain the compound of formula 12.
In the present invention, the second condensation reaction: 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: the molar ratio of the compound having a structure represented by formula 10 to the compound having a structure represented by formula 11 is preferably 1 (1.1 to 1.3), more preferably 1. The molar ratio of the compound having a structure represented by formula 10 to the condensation reagent is preferably 1.2. The molar ratio of the compound having a structure represented by formula 10 to the dehydrating agent is preferably 1.1. The amount of the organic base reagent is preferably adjusted to a pH value of 8 to 9 in 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 agent, the dehydrating agent and the organic solvent. The invention has no special requirement on the dosage of the organic solvent, and the second condensation reaction is ensured to be smoothly carried out.
In the present invention, the second condensation reaction: 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 a formula 10; under the condition of ice-water bath, 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 to obtain a first mixed solution; mixing the first mixed solution and a compound with the structure shown in formula 11 to obtain a second mixed solution; and dropwise adding the organic alkali reagent into the second mixed solution, adjusting the pH value of the second mixed solution to 8-9, and withdrawing the mixed solution from the ice water bath.
In the present invention, the temperature of the second condensation reaction is preferably room temperature. The time for the second condensation reaction is preferably overnight. The second condensation reaction is carried out under stirring. According to the invention, the second condensation reaction is preferably detected by TLC, and the developing solvent used by TLC is preferably CH 2 Cl 2 /CH 3 OH,v:v＝20/1。
In the present invention, the second condensation reaction is carried out to obtain a second condensation reaction solution, and in the present invention, it is preferable to carry out a post-treatment of 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 residue and ethyl acetate, and then carrying out solid-liquid separation to remove DCU generated after DCC participates in the reaction, thereby obtaining 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 present invention preferably removes impurities by solid-liquid separation. The washing is preferably: saturated NaHCO was performed sequentially 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, and washing with saturated KHSO 4 Washing with an aqueous solution; each solution is preferably washed 3 times each. In the present invention, the drying is preferably performed overnight using anhydrous sodium sulfate. In the present invention, a specific embodiment of the solvent removal is preferably concentration under reduced pressure.
After obtaining a compound having a structure represented by formula 12, the present invention mixes the compound having a 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 a compound having a structure represented by formula 5.
In the present invention, in the deprotection reaction of the first amino group, 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 having a structure represented by formula 12 to the volume of the organic solvent for hydrogen chloride is preferably 1g.
In the present invention, the temperature of the first amino deprotection reaction is preferably room temperature, the time of the first amino deprotection reaction is preferably 4h, and the first amino deprotection reaction is preferably performed under stirring. The first amino deprotection reaction is preferably detected by TLC, and a developing agent used by TLC is preferably CH 2 Cl 2 /CH 3 OH,v:v＝20/1。
In the present invention, the first amino group deprotection reaction gives a first deprotection reaction solution, and in the present invention, it is preferable to perform a post-treatment on the first deprotection reaction solution to give a compound having a structure represented by formula 5. In the present invention, the post-treatment preferably comprises: the first deprotection reaction solution was first pumped to dryness under reduced pressure using a water pump, and then washed 3 times with 20ml of anhydrous ethyl acetate. In the present invention, in the amide condensation reaction, the condensation reagent 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 amide condensation reaction: the molar ratio of the compound having a structure represented by formula 4 to the compound having a structure represented by formula 5 is preferably 1 (1.1 to 1.3), more preferably 1. The molar ratio of the compound having the structure represented by formula 4 to the condensing agent is preferably 1.2. The molar ratio of the compound having a structure represented by formula 4 to the dehydrating agent is preferably 1.1. The amount of the organic base reagent is preferably adjusted to a pH value of 8 to 9 in a mixed solution obtained by mixing a compound having a structure represented by formula 4, a compound having a structure represented by formula 5, a condensation reagent, a dehydrating agent and an organic solvent. The invention has no special requirement on the dosage of the organic solvent, and the amide condensation reaction is ensured to be smoothly carried out.
In the present invention, the amide condensation reaction: 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 a formula 4; under the condition of ice-water bath, 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 to obtain a first mixed solution; mixing the first mixed solution and a compound with the structure shown in the formula 5 to obtain a second mixed solution; and dropwise adding the organic alkali reagent into the second mixed solution, adjusting the pH value of the second mixed solution to 8-9, and withdrawing the mixed solution from the ice water bath.
In the present invention, the temperature of the amide condensation reaction is preferably room temperature. The time for the amide condensation reaction is preferably overnight. The amide condensation reaction is carried out under stirring. According to the invention, the amide condensation reaction is preferably detected by TLC, and the developing solvent used by TLC is preferably CH 2 Cl 2 /CH 3 OH,v:v＝20/1。
In the present invention, the amide condensation reaction produces an amide condensation reaction liquid, and it is preferable to subject the amide condensation reaction liquid to a post-treatment to produce 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 residue 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 impurities are preferably removed by the solid-liquid separation in the present invention. The washing is preferably: saturated NaHCO was performed sequentially 3 Washing with aqueous solution, washing with saturated NaCl aqueous solution, and washing with saturated KHSO 4 Washing with an aqueous solution; each solution is preferably washed 3 times each. In the present invention, the drying is preferably carried out overnight using anhydrous sodium sulfate. In the present invention, a specific embodiment of the solvent removal agent is preferably concentrated under reduced pressure.
After obtaining the compound having the structure represented by formula 6, the present invention mixes the compound having the structure represented by formula 6 with an organic solution of hydrogen chloride to perform an amino group deprotection reaction (hereinafter referred to as a second amino group deprotection reaction) to obtain a compound having 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 having the structure represented by formula 6 to the volume of the organic solvent of hydrogen chloride is preferably 1 g.
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 4h, and the second amino deprotection reaction is preferably performed under stirring. The second amino deprotection reaction is preferably detected by TLC (thin layer chromatography), and a developing agent used by TLC is preferably CH 2 Cl 2 /CH 3 OH,v:v＝20/1。
In the present invention, the second amino deprotection reaction yields a second deprotection reaction solution, and in the present invention, it is preferable to perform post-treatment on the second deprotection reaction solution to yield a compound having a structure represented by formula 2. In the present invention, the post-treatment preferably comprises: the second deprotection reaction solution was first pumped to dryness under reduced pressure using a water pump, and then washed 3 times with 20ml of anhydrous ethyl acetate. In the present invention, the coupling reagent is preferably O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU) during the coupling reaction. The organic solvent is preferably N' N-Dimethylformamide (DMF). The molar ratio of the loganic acid to the compound with the structure shown in the formula 2 is preferably 1 (1.9-2.2), and more preferably 1 (2-2.15). The mass ratio of the loganic acid to the coupling agent is preferably 1. The invention has no special requirement on the dosage of the organic solvent, and the coupling reaction is ensured to be smoothly carried out.
In the present invention, the mixing is preferably performed at the time of the coupling reaction: mixing the loganic acid, the compound with the structure shown in the formula 2, the organic solvent and the coupling reagent in sequence.
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 present invention, the coupling reaction is performed to obtain a coupling reaction solution, and in the present invention, the coupling reaction solution is preferably post-treated to obtain a compound having a structure represented by formula 3. In the present invention, the post-treatment preferably comprises: removing the solvent from the coupling reaction solution to obtain a solid residue; fixing the aboveAnd (4) separating the residue 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 chromatography separation preferably adopts reversed phase C 18 The eluent is preferably a mixed solvent of water and acetonitrile, and the gradient elution is as follows: water: and (v) acetonitrile, wherein the volume content of the acetonitrile is changed in a gradient manner of 0% → 50%, the total elution time is preferably 20min, the peak time is about 15min, the eluent is removed after the eluent is obtained, and the specific implementation mode of removing the eluent is preferably 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, a palladium-carbon catalyst and an organic solvent are mixed in a hydrogen atmosphere to carry out reduction deprotection reaction, and the maleic acid derivative 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 having a structure represented by formula 3 to the palladium on carbon catalyst is preferably 1.1. The invention has no special requirement on the dosage of the organic solvent, and ensures that the reduction deprotection reaction is smoothly carried out.
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 obtains a reduction deprotection reaction solution, and the invention preferably carries out post-treatment on the reduction deprotection reaction solution to obtain the loganic acid derivative with the structure shown in the formula 1. In the present invention, the post-treatment preferably comprises: removing part of solvent from the reduction deprotection reaction solution to obtain a humidity solid residue; and (3) separating the wet solid residue by adopting column chromatography to obtain the loganic acid derivative 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 chromatography separation preferably adopts reversed phase C 18 The eluent is preferably a mixed solvent of aqueous formic acid and acetonitrile, and the gradient elution is as follows: 0.5wt% formic acid aqueous solutionLiquid: and (v) acetonitrile, wherein the volume content of the acetonitrile is changed in a gradient manner of 0% → 20%, 0-20%, the total elution time is 20min, the peak time is about 10min, the eluent is removed after the eluent is obtained, and the specific implementation mode of the eluent removal is preferably rotary evaporation.
The invention provides application of the loganin acid derivative in the technical scheme or the loganin acid derivative prepared by the preparation method in the technical scheme in preparation of a medicine for treating and/or preventing the Parkinson nervous system degenerative disease or disorder.
For further explanation of the present invention, the technical solutions provided by the present invention are described in detail below with reference to the drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A loganic acid derivative of the structure shown in formula 1 was prepared according to the schemes shown in figures 1 and 2:
3.05g (10 mmol) of a compound (Boc-Arg (NO) with the structure shown in formula 7 2 ) Suspended in 100-120 mL of anhydrous tetrahydrofuran to obtain Boc-Arg (NO) 2 ) Suspending the solution, and adding Boc-Arg (NO) under ice-bath condition 2 ) 1.62g (12 mmol) of 1-hydroxybenzotriazole (HOBt) was added to the solution, and then 2.27g (11 mmol) of Dicyclohexylcarbodiimide (DCC) was added thereto, followed by stirring for 20 to 30min to obtain a first mixed solution.
Adding 2.42g (12 mmol) of the compound (Gly-OBzl & HCl) with the structure shown in the formula 8 into the first mixed solution under ice bath to obtain a second mixed solution, dropwise adding 3-4 mL of N-methylmorpholine to adjust the pH value of the second mixed solution to 9, removing the ice bath, and stirring at room temperature overnight. TLC monitoring of the reaction (CH) 2 Cl 2 /CH 3 OH, v: v = 20/1), the reaction solution obtained after the reaction was concentrated under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, and dicyclohexylurea (DCU, an impurity generated after the reaction was completed by DCC) was filtered off. Taking the filtrate and sequentially using saturated NaHCO 3 Washing with an aqueous solution (12 mL. Times.3), washing with a saturated aqueous NaCl solution (12 mL. Times.3), and saturating KHSO 4 Aqueous wash (12 mL. Times.3), saturated aqueous NaCl wash (12 mL. Times.3), saturated aqueous NaHCO 3 Washing with aqueous solution (12 mL. Times.3), and dissolving with saturated NaCl solutionWash with liquid (12 mL. Times.3). The washed ethyl acetate phase was dried over anhydrous sodium sulfate overnight. Filtering, and concentrating the filtrate under reduced pressure to obtain 4g white foam, which is compound (Boc-Arg (NO) with structure shown in formula 9 2 )-Gly-OBzl)。
2.5g of a compound having the structure described in formula 9 (Boc-Arg (NO) 2 ) Dissolving (Boc-Arg (NO) with 10-12 mL of methanol to obtain (Gly-OBzl) 2 ) -Gly-OBzl) solution; then (Boc-Arg (NO) was treated with 2-3 mL of 2N NaOH aqueous solution under ice-bath conditions 2 ) Adjusting the pH value of the solution of-Gly-OBzl) to 13-14, stirring for 3.5-4 h, and monitoring the completion of the reaction (CH) by TLC 2 Cl 2 /CH 3 OH, v: v = 20/1). After the hydrolysis reaction is finished, 3-4 mL of saturated KHSO is added into the mixture obtained by the reaction under the ice-bath condition 4 The pH was adjusted to 7 and the methanol was removed by concentration under reduced pressure. The residue obtained by concentration under reduced pressure in an ice bath was redissolved with pure water and then 3mL of saturated KHSO was added 4 The pH was adjusted to 2 and redissolved with ethyl acetate, extracted with ethyl acetate (12 mL. Times.3), and the combined ethyl acetate extracts were dried over anhydrous sodium sulfate overnight. Filtering, and concentrating the filtrate under reduced pressure to obtain about 2g white solid, i.e. compound (Boc-Arg (NO) with structure shown in formula 4) 2 )-Gly)。
Dissolving 3.23g (10 mmol) of a compound (Boc-Asp (OBzl)) with the structure shown in the formula 10 in 100-120 mL of anhydrous tetrahydrofuran to obtain a Boc-Asp (OBzl) solution, adding 1.62g (12 mmol) of 1-hydroxybenzotriazole (HOBt) into the Boc-Asp (OBzl) solution under ice bath conditions, adding 2.27g (11 mmol) of Dicyclohexylcarbodiimide (DCC), and stirring for 20-30 min to obtain a third mixed solution;
adding 4.6g (12 mmol) of compound (Val-OBzl. Tos) with the structure shown in formula 11 into the third mixed solution under ice bath condition to obtain a fourth mixed solutionAnd 3-4 mL of N-methylmorpholine is added dropwise to adjust the pH value of the fourth solution to 9, the ice bath is removed, and the fourth solution is stirred at room temperature overnight. TLC monitoring of the reaction (CH) 2 Cl 2 /CH 3 OH, v: v = 20/1), concentrating the reaction solution under reduced pressure after the reaction is completed, dissolving the residue with 100mL of ethyl acetate, and filtering off DCU. The filtrate was sequentially treated with saturated NaHCO 3 Washing with an aqueous solution (12 mL. Times.3), washing with a saturated aqueous NaCl solution (12 mL. Times.3), and saturating KHSO 4 Aqueous wash (12 mL. Times.3), saturated aqueous NaCl wash (12 mL. Times.3), saturated aqueous NaHCO 3 An aqueous solution (12 mL. Times.3) and a saturated aqueous NaCl solution (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, i.e., the compound represented by formula 12 (Boc-Asp (OBzl) -Val-OBzl).
4g of the compound having the structure represented by formula 12 (Boc-Asp (OBzl) -Val-OBzl) was slowly dissolved in 40mL of an ethyl acetate solution of hydrogen chloride at a hydrogen chloride molar concentration of 4mol/L under ice-bath conditions. Boc-Asp (OBzl) -Val-OBzl solution was obtained, stirred for 4h 2 Cl 2 /CH 3 OH, v: v = 20/1). And (3) concentrating the reaction liquid obtained after the reaction under reduced pressure by using a water pump, re-dissolving the residue by using 20mL of anhydrous ethyl acetate, concentrating under reduced pressure, and repeating for 3 times to obtain a yellow oily substance, namely the compound (Asp (OBzl) -Val-OBzl) with the structure shown in the formula 5.
10mmol of a compound (Boc-Arg (NO)) having a structure represented by the formula 4 2 ) -Gly) is dissolved in 100-120 mL of anhydrous tetrahydrofuran to obtain Boc-Arg (NO) 2 ) -Gly solution under ice-bath condition to Boc-Arg (NO) 2 ) 1.62g (12 mmol) of 1-hydroxybenzotriazole (HOBt) was added to the-Gly solution, and then 2.27g (11 mmol) of Dicyclohexylcarbodiimide (DCC) was added thereto, followed by stirring for 20 to 30min to obtain a fifth mixed solution.
And adding 12mmol of the compound (Asp (OBzl) -Val-OBzl) with the structure shown in the formula 5 into the fifth mixed solution under ice bath to obtain a sixth mixed solution, dropwise adding 3-4 mL of N-methylmorpholine to adjust the pH value of the sixth mixed solution to 9, removing the ice bath, and stirring at room temperature overnight. TLC monitoring of the reaction (CH) 2 Cl 2 /CH 3 OH, v: v = 20/1), the reaction solution obtained after the completion of the reaction was concentrated under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, and dicyclohexylurea (DCU, an impurity generated after the reaction was completed by DCC) was filtered off. Taking the filtrate and sequentially using saturated NaHCO 3 Washing with an aqueous solution (12 mL. Times.3), washing with a saturated aqueous NaCl solution (12 mL. Times.3), and saturating with KHSO 4 Aqueous wash (12 mL. Times.3), saturated aqueous NaCl wash (12 mL. Times.3), saturated aqueous NaHCO 3 The resulting mixture was washed with an aqueous solution (12 mL. Times.3) and with a saturated aqueous NaCl solution (12 mL. Times.3). The washed ethyl acetate phase was dried over anhydrous sodium sulfate overnight. Filtering, and concentrating the filtrate under reduced pressure to obtain light yellow solid (Boc-Arg (NO) with structure shown in formula 6) 2 ) -Gly-Asp (OBzl) -Val-OBzl) in 55% yield. The mass spectrum of the compound having the structure shown in formula 6 is shown in fig. 3, and the nuclear magnetic hydrogen spectrum data is shown in fig. 5.
4g of a compound having a structure represented by formula 6 (Boc-Arg (NO) was added under ice-bath conditions 2 ) -Gly-Asp (OBzl) -Val-OBzl) was slowly dissolved in 40mL of hydrogen chloride in ethyl acetate at a molar concentration of 4mol/L. Obtaining Boc-Arg (NO) 2 ) -Gly-Asp (OBzl) -Val-OBzl solution, stirred for 4h 2 Cl 2 /CH 3 OH, v: v = 20/1). Concentrating the reaction solution under reduced pressure with water pump, re-dissolving the residue with 20mL anhydrous ethyl acetate, concentrating under reduced pressure, and repeating for 3 times to obtain white powder, which is Arg (NO) compound with structure shown in formula 2 2 ) -Gly-Asp (OBzl) -Val-OBzl) in 56.6% yield.
Under the ice-bath condition, 1g (2.6 mmol) of loganin acid and 1.21g (5.3 mmol) of compound (Arg (NO) with the structure shown in the formula 2 ) -Gly-Asp (OBzl) -Val-OBzl) is dissolved in 25-30mLN' N-Dimethylformamide (DMF), 1.2g O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU) is added for reaction at room temperature overnight, the obtained reaction liquid is dried in a spinning mode, and the residue is subjected to reversed phase C 18 Purifying the column under the following conditions: inverse phase C 18 And the eluent is a mixed solvent of water and acetonitrile, and the ratio of water: acetonitrile (v: v), wherein the volume content of the acetonitrile is changed in a gradient manner of 0% → 50%, the total elution time is 20min, the peak emergence time is about 15min, and the flow rate of the eluent is 50mL/min; spin-drying to obtain white powder, i.e. compound (loganin-Arg (NO) with structure shown in formula 3 2 ) -Gly-Asp (OBzl) -Val-OBzl) in 26% yield.
Adding a compound with a structure shown in a 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 the formula 3 to the palladium carbon catalyst is 1) into 8-10 mL of methanol, stirring, introducing hydrogen for one night, filtering, removing most of a solvent by spinning, but not drying by spinning, and using a reversed phase C 18 Preparation of the column a white powder was obtained with the conditions: c 18 And the eluent is: 0.5wt% aqueous formic acid and acetonitrile, 0.5wt% aqueous formic acid: acetonitrile (v: v), gradient elution: 0.5wt% aqueous formic acid: and (v) acetonitrile (v, v), wherein the volume content of the acetonitrile is changed in a gradient manner of 0% → 20%, 0-20%, the total elution time is 20min, the peak time is about 10min, partial salts are likely to be formed, and the loganic acid derivative with the structure shown in the formula 1 is obtained, and the yield is 42%. The mass spectrum of the loganin acid derivative with the structure shown in the formula 1 is shown in figure 4, and the hydrogen nuclear magnetic spectrum is shown in figure 6. The UV spectra of loganic acid and loganic acid derivatives of the structure shown in formula 1 are shown in FIG. 7.
Test example
2. Examples of biological Activity
2.1 Experimental drugs
Test agent LA-1 (a loganic acid derivative of formula 1 prepared in example 1)
2.2 Experimental reagents
Ceramide standard (860517), sphingomyelin standard (860584) (Avanti Polar Lipids, alabama, USA); chloroform chromatographically pure (11310278) (Fisher Scientific, pittsburgh, PA, USA); 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (M0896), methanol chromatographically pure (34860), pure ammonia (5.43830), ethanol (1012768) (Sigma, st. Louis, MO, USA); ready-to-use immunohistochemistry KIT (KIT-5001) (Maxim, fuzhou, china); TUNEL apoptosis kit (KGA 7061) (KeyGEN Biotech, nanking, china); mouse Dopamine (DA) ELISA Kit (CSB-E08661 m) (Cusabio, houston, USA); mouse 3,4 dihydroxyphenylacetic acid (Mouse DOPAC) ELISA kit (MBS 7269842) (Mybiosource, san Diego, USA); homovanillic Acid (HVA) ELISA Kit (abx 150352) (Abbexa, cambridge, UK); 5-HT ELISA Kit (ab 285243), mouse IL-1beta antibody (ab 197742) (Abcam, cambridge, UK); mouse TNF-alpha antibody (EM 0183) (Finetest, hubei, china); superoxide dismutase (SOD) assay kit (A001-3) (Nanjing Bioengineering Institute, nanjing, china); glutathione peroxidase assay kit (BC 1195) (Solarbio, beijing, china); malondialdehyde (MDA) assay kit (S0131S) (Beyotime, shangHai, china); anti-PSD-95antibody (3450) available from CST, inc. (Cell Signaling Technology, boston, USA); anti-TH antibodies (ab 137869), anti-SYN antibodies (ab 52636) available from Abcam corporation (Abcam, cambridge, UK); mouse anti-rabbitIgG-FITC antibody (sc-2359) was purchased from Santa Cruz, inc. (Santa Cruz, calif., USA).
Note: the operation of the kit of the present invention is described with reference to the kit instructions.
2.3 Experimental instruments
A FORMA 700 model ultra-low temperature refrigerator, thermo corporation; YC-300L type drug storage cabinet, miao national Mitsubishi low temperature science and technology, inc.; direct-Q withpump type ultrapure water instrument, millipore corporation; SWCJ-2FD type superclean bench: suzhou clarification plant, inc.; 3K15 model low temperature high speed centrifuge, sigma company; BS224 type electronic balance: beijing Saedodes Instrument systems, inc.; paraffin embedding machine, microtome, LEICA company, germany; berthold LB941 micropore plate type multifunctional microplate reader, berthold company; olympus inverted phase contrast microscope, olympus corporation, japan; zeiss LSM laser confocal microscope, zeiss company, germany; labmazeV3.0 animal behavior trajectory analysis System, beijing Zhongguo Teddy development Co., ltd; ZS-KC type open field case, beijing Zhongguo Dichu science and technology development Co., ltd; mouse pole climbing experimental device, jiangsu seon biotechnology limited; 1260 high performance liquid chromatograph, agilent, usa; 6410B triple quadrupole tandem mass spectrometer, agilent, USA.
2.4 Experimental animals
Female C57BL/6 mice, 8 weeks old; body weight 20 ± 2g, provided by changzhou kavens laboratory animals ltd, animal certification No.: SCXK 2021-0010, raised in 22 + -2 deg.C environment, and taken freely and drunk freely.
2.5 Experimental methods
2.5.1 mouse model construction
After C57BL/6 mice are adaptively raised for one week, MPTP is injected into the abdominal cavity every day, the injection dose is 30mg/kg,
the injection volume was 0.1mL/10g, MPTP (Parkinson's disease molding reagent) was daily recorded as Day0, and the injection was continued for 5 days.
2.6 Experimental groups
Normal group: c57BL/6 mice were normally housed, and were administered once daily to Ddy (N = 6) by tail vein injection of an equal volume of saline beginning with Day 5;
model team (MPTP team): after the PD model of the C57BL/6 mouse is modeled, equal volume of physiological saline is injected into the tail vein of the Day5, and the medicine is continuously administered to Ddy14 (N = 6) once a Day;
MPTP + LA-1 group: after the model building of the PD model of the C57BL/6 mouse is finished, LA-1 (the injection dose is 8.04 mg/kg) is continuously administered to Ddy14 (N = 6) by feeding LA-1 once a Day in the tail vein of Day5, wherein the injection volume is 0.1 mL;
performing behavioural experiments such as open field experiments, pole climbing experiments and the like 12 hours after the last administration; after the behavioral experiments, CO 2 Euthanizing the mice by a method, taking the substantia nigra compact part of the brain of each group of mice, and detecting the TH content in tissue samples of each group of mice by an immunohistochemical experiment; TUNEL staining to detect DA-competent neuronal cell apoptosis in each group of mouse tissues; detecting the content of DA, DOPAC, HVA, 5-HT, IL-1beta and TNF-alpha in mouse tissues by ELISA; detecting protein expression of SYN and PSD-95 in substantia nigra region in a mouse tissue section by an immunofluorescence experiment; the kit detects the contents of SOD, MDA and GSH-Px in the mouse brain black matrix area; and detecting the contents of sphingomyelin and ceramide in the substantia nigra region by an LC-MS/MS experiment.
2.7 open field experiment
The ZS-KC open field is used for observing and researching the neuropsychiatric changes of experimental animals and various behaviors after the experimental animals enter an open environment, and the size of a mouse activity open field box in the experiment is 500 multiplied by 350mm. The peripheral lattices are called along the wall lattices, and the other lattices are central lattices. Each animal was placed in the center bottom grid and all the movement trajectories and behavioral parameters of the mice were collected using a camera and Labmaze V3.0 animal behavior trajectory analysis system.
Before the experiment, each strain of mouse is adapted to a feeding room for 1 week, and the mouse is grabbed for about 5min every day during the period to adapt to the operation of an experimenter; the mice were moved into the testing room 30min before the start of the experiment to adapt, so as to ensure that the activity of the mice tends to be stable during the experiment. The mouse is placed in the center of an open field with the back facing the operator, the camera observes and records the activity within 5min, and the track of the mouse is recorded by using a camera and a Labmaze V3.0 animal behavior track analysis system. And (3) clearing excrement of the bottom plate of the open box after the experiment of each animal is finished, spraying 500mL/L ethanol on the bottom of the open box, and wiping the excrement by clean gauze to prevent the residual smell of the animal of the previous experiment from influencing the next experiment.
2.8 Pole climbing experiment
Holding the tail of the mouse, placing the head end of the mouse downwards on the rod top, enabling four limbs of the mouse to grasp the rod top, recording the total time of the mouse from the beginning of the movement of climbing from the rod top to the rod bottom to the landing of all the limbs of the mouse, recording the time, analyzing and comparing to detect the action coordination of the mouse, and recording the gait abnormity in the climbing process. The mice were guided through this behavioural experiment before testing, three times per mouse, each time at 5min intervals, and the mean value was taken.
2.9 immunohistochemistry
1) Baking slices: placing the prepared paraffin section in an electric heating constant-temperature drying box, and baking for 3 hours at 60 ℃;
2) Carrying out conventional xylene dewaxing on the dried paraffin sections, carrying out downward gradient ethanol hydration, and washing with distilled water;
3) Antigen retrieval;
4) Dropwise addition of 3wt% of 2 O 2 Incubating at room temperature for 10min to inactivate endogenous enzymes, washing with PBS for 3 times, each for 3min;
5) Adding normal goat immune serum dropwise to each section, sealing, incubating at room temperature for 10min, removing excess liquid, washing, adding TH antibody dropwise (TH antibody is diluted at a ratio of 1;
6) Taking out from refrigerator, recovering to room temperature, washing with PBS for 3 times, each for 3min;
7) Dripping polymer intensifier into each slice, washing at room temperature for 20min, washing with PBS for 3 times, each for 3min;
8) Dripping enzyme-labeled anti-mouse rabbit polymer into each slice, washing for 3 times (3 min each time) at room temperature for 10min with PBS;
9) DAB color development, controlling reaction time under a microscope, performing hematoxylin counterstain, and fully washing with distilled water to stop color development;
10 Slice is dehydrated and dried by gradient alcohol, xylene is transparent, and neutral gum is sealed;
11 Pictures were taken using a phase contrast microscope at 400 x field of view.
2.10TUNEL staining
1) Paraffin section is dewaxed by a conventional method, and the section is dried at 60 ℃ for 60min. Xylene dewaxing 2 times, ethanol hydration (100%, 95%, 80%, 75%); 5min each time;
2) The slices were rinsed three times for 5min each time by immersion in 1 × PBS;
3) Preparing a protease K working solution: the number of the calculated samples is intensively prepared, and 10 mu L of 10 XProteinase K is added into 90 mu L of 1 XPBS of each sample, and the samples are prepared immediately after use;
4) 100 mu.L of protease K working solution is dripped on each tissue slice, and the reaction is carried out for 30min at 37 ℃. Rinsing the slices with 1 × PBS for three times, 5min each time;
5) According to the type of the sample, 100. Mu.L of DNase I reaction solution containing different activity units was prepared according to the method shown in Table 1:
TABLE 1 preparation method of DNase I reaction solution
| Sample(s) | Paraffin section |
| Μ/100μL | 3000～5000Μ |
| DNase I (50 μm/μ L) |
60～100μL |
| DNase |
40～0μL |
6) 100 μ L of DNase I (deoxyribonuclease I) reaction solution prepared according to Table 1 was dropped on a sample piece, treated at 37 ℃ for 30min,
7) Immersing the positive plate in 1 × PBS for rinsing three times, 5min each time;
8) Preparing a TdT enzyme reaction solution: the number of samples is calculated and centralized preparation is carried out (negative is not counted in the photos), and the dosage of each sample is as follows: add 1.0. Mu.L of biotin-11-d. Mu.M TP and 4.0. Mu.L of TdT Enzyme to 45. Mu.L of emulsification Buffer, ready to use, and keep out of the light;
9) The sample periphery was blotted with absorbent paper, 50. Mu.L of TdT enzyme reaction solution was dropped on each sample, and the mixture was put into a incubator and reacted for 60min at 37 ℃ in the dark. (Note: negative control sample without TdT enzyme reaction solution)
10 The reacted sample piece is immersed in 1 XPBS for rinsing three times, 5min each time, and the sample piece is protected from light;
11 Streptavidin-TRITC reagent was mixed with 45. Mu.L of labelling Buffer at 5. Mu.L per section, and the total amount was calculated, i.e., ready to use, and carefully protected from light.
12 ) the sample is sucked dry by absorbent paper, 50. Mu.L of Streptavidin-TRITC labeling solution is dropped on each sample, and the mixture is put into a wet box and reacted for 30min at 37 ℃ in the dark.
13 The reacted sample piece is immersed in 1 XPBS for rinsing three times, 5min each time, and the sample piece is protected from light;
14 DAPI staining solution to counterstain the cell nucleus, and reacting for 10min at room temperature in a dark place. The DAPI staining solution was washed away and an appropriate volume ratio of mounting medium (glycerol: PBS = 6:4) was added.
15 Fluorescence microscopy (630 ×): excitation wavelength 543nm and emission wavelength 571nm; (Note: fluorescence is easily quenched, please observe the picture as soon as possible).
2.11 immunofluorescence
1) Washing the mouse brain tissue section with PBS for 5min and 3 times;
2) Wiping off liquid around the sliced tissues, and utilizing the hydrophobic effect of an immunohistochemical pen to draw a circle at a position 2mm away from the tissues so as to prevent various liquid components dripped after dilution;
3) 0.4 percent TritonX-100 percent, incubating and soaking for 10min at room temperature; washing with PBS for 5min × 3 times;
4) Antigen retrieval (cold water pressure reduction 1.5min after pressure cooker pressure limiting valve gassing). Naturally cooling, washing with PBS for 5min × 3 times;
5) Sealing 5% sheep serum at 37 deg.C for 10min;
6) The serum was discarded and primary antibody (SYN antibody diluted at 1; the PSD-95antibody was diluted at a ratio of 1;
7) The antibody reaction solution was aspirated and washed with PBS for 5min × 3 times;
8) Dripping a fluorescence-labeled secondary antibody (mouse anti-rabbitIgG-FITC antibody is diluted according to the proportion of 1;
9) The antibody reaction solution was aspirated and washed with PBS for 5min × 3 times;
10 ) 90% glycerol seal was placed in a dark box;
11 630 x field of view of confocal laser microscopy.
2.12 detection of DA content in mouse black zone tissue by ELISA
1) The tissue of the black zone of the mice was washed with 1 XPBS and the homogenate was stored in 1 XPBS overnight at-20 ℃. After membrane rupture through twice freeze-thaw cycles, the homogenate is centrifuged for 5min at 2-8 ℃ at 5000 Xg. The supernatant was immediately removed and assayed.
2) Calculating and determining the number of pre-coated laths required by one experiment, taking out the required laths, placing the laths in a 96-hole frame, temporarily sealing the laths by putting back an aluminum foil bag, and storing at 4 ℃.
3) Setting a blank hole, a standard product hole and a sample hole, and respectively adding a sample or standard products with different concentrations into the corresponding holes according to 50 mul/hole, wherein the concentrations of the standard products are as follows: 1000pg/mL, 250pg/mL, 80pg/mL, 20pg/mL, 5pg/mL, 0pg/mL.
4) In addition to blank wells, 50. Mu.L of HRP-conjugate was added per well. Add 50. Mu.L of antibody to each well, mix well, incubate for 1h at 37 ℃.
5) The liquid was discarded from each well and the washing was repeated 3 times.
6) Add 50. Mu.L each of Substrate A and Substrate B to each well, mix well, and incubate at 37 ℃ for 15min.
7) Add 50. Mu.L of stop solution per well and immediately record the read at 450 nm.
2.13ELISA detection of DOPAC content in mouse melanotic zone tissues
1) Tissue homogenization: in this experiment, the tissue was rinsed in ice cold with PBS to completely remove excess blood. The tissue was minced and homogenized in 500. Mu.L PBS using an ice-filled glass homogenizer. The resulting suspension was sonicated to further disrupt the cell membrane. The homogenate was then centrifuged at 1500 Xg for 15min and the supernatant was taken for experimentation.
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 substance concentration is 10ng/mL, 5ng/mL, 2.5ng/mL, 1.0ng/mL, 0.5ng/mL.
3) Add 50. Mu.L of Conjugate to each well (except blank control well) and mix well; sealing plates, and incubating for 1h at 37 ℃;
4) Discarding liquid in each hole, and washing the plate for 5 times;
5) Add 50. Mu.L of Substrate A and Substrate B to each well, respectively; sealing plates, and incubating for 20min at 37 ℃;
6) Add 50. Mu.L of Stop Solution per well and immediately record the read at 450 nm.
2.14 detection of HVA content in mouse black zone tissue by ELISA
1) Tissue homogenization: the tissues were rinsed with ice cold PBS and excess blood was removed. The tissue was minced and placed 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 well, a control well and a standard well, and adding 50 mu L of a sample, a standard dilution buffer or a standard into the sample well, the control well and the standard well respectively; the standard substance concentration is 200ng/mL, 66.7ng/mL, 22.2ng/mL, 7.41ng/mL, 2.47ng/mL.
3) Add 50. Mu.L Detection Reagent A to each well, incubate 1h at 37 ℃;
4) Discarding liquid in each hole, and washing for 3 times;
5) Add 100. Mu.L Detection Reagent B to each well, incubate 30min at 37 ℃;
6) Discarding liquid in each hole, and washing for 5 times;
7) Adding 90 μ L of TMB base solution into each well, and incubating at 37 deg.C for 20min;
8) Add 50. Mu.L of stop solution per well and immediately record the read at 450 nm.
2.15ELISA detection of 5-HT content in mouse black zone tissue
1) Tissue homogenization: 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 disrupt the tissue cells, the suspension is sonicated with an ultrasonic cell disrupting agent. 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, sealed with an aluminum foil bag until the strips were temporarily removed, and stored at 4 ℃.
3) Setting a standard hole, a sample hole and a control hole; respectively adding the samples or standard substances with different concentrations into corresponding holes according to the concentration of 50 mu L/hole, wherein the standard substance concentration is as follows: 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 0ng/mL; add 50. Mu.L of Biotin-detection antibody purification solution immediately to each well, mix well and incubate at 37 ℃ for 45min.
4) The liquid from each well was discarded and washed 3 times.
5) Add 0.1mL of SABC working solution per well, seal plate, incubate 30min at 37 ℃.
6) The liquid from each well was discarded and washed 5 times.
7) mu.L of TMB substrate was added to each well, and the wells were sealed and incubated for 30min at 37 ℃ in the absence of light.
8) Add 50. Mu.L of Stop solution per well and immediately record the read at 450 nm.
2.16ELISA detection of IL-1beta content in mouse black zone tissue
1) Tissue homogenization: tissue was first minced and thoroughly rinsed in PBS to remove blood at a rate of 500 μ L of 1 × frozen cell extraction buffer per 100mg of wet tissue, and the volume of extraction buffer added was adjusted accordingly. Incubate on ice for 20min. Centrifuging at 18000 Xg for 20min at 4 deg.C; taking the supernatant for detection.
2) Calculating and determining the number of pre-coated laths required by one experiment, taking out the required laths, placing the laths in a 96-hole frame, temporarily sealing the laths by putting back an aluminum foil bag, and storing at 4 ℃.
3) Setting a blank hole, a standard hole and a sample hole, wherein the standard concentration 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 of Antibody Cocktail was added to each well, the reaction wells were sealed with a sealing membrane, and incubated for 60min at room temperature in a 400rpm plate shaker.
5) Each well plate was washed 3 times with 350 μ L of wash buffer, completely removing liquid after each step, and after the last wash the plate was inverted and smeared on a clean paper towel to remove excess liquid.
6) Add 100. Mu.L of color reagent TMB solution to each well, seal the reaction well with a sealing plate (white), and incubate in a shaker at 400rpm for 10min in the dark at room temperature. When the room temperature is lower, the incubation time needs to be prolonged appropriately, and the incubation can be carried out until the standard substance and the sample have a very remarkable color change.
7) mu.L of stop solution was added to each well, and the OD at 450nm was recorded.
2.17ELISA detection of TNF-alpha content in mouse black zone tissue
1) Tissue homogenization: and (3) washing the tissue of the mouse brain black matrix area by using precooled PBS buffer solution, and removing residual blood. The tissue was weighed, minced, and homogenized on ice using a glass homogenizer. To further disrupt the tissue cells, the suspension may be sonicated with an ultrasonic cell disruptor. The homogenate was then centrifuged at 5000 Xg for 5min to give a supernatant.
2) Setting blank holes, standard product holes and sample holes, and adding 100 mu L of sample and standard product into each hole respectively; the concentration of the standard substance is as follows: 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.625pg/mL, 7.812pg/mL, 3.906pg/mL, 0pg/mL.
3) Sealing the plate, and incubating for 90min at 37 ℃;
4) Discarding liquid in each hole, and washing the plate twice;
5) Adding 100 μ L Biotin-labeled antibody purification solution into each well, sealing the plate, and incubating at 37 deg.C for 90min;
6) Discarding liquid in each hole, and washing the plate for three times;
7) Adding 90 mu L of TMB Substrate into each hole, sealing the plate, and incubating for 20min at 37 ℃ in a dark place;
8) Add 50. Mu.L of Stop Solution to each well and record the OD at 450 nm.
2.18 kit for detecting SOD content in mouse brain black zone
1) Tissue homogenization: homogenizing tissue by using PBS, wherein the weight of the tissue accounts for 10% of that of the homogenate or lysate; after homogenization or lysis, the supernatant was centrifuged at 2500rpm for 10min for subsequent assays.
2) Reagents were added according to Table 2
TABLE 2 kinds and amounts of reagents
2.19 kit for detecting MDA content of mouse brain black matter area
1) Tissue homogenization: the tissue was homogenized with PBS, as indicated in table 4 and table 5 for the type and amount of reagents, with a tissue weight of 10% of the homogenate or lysate; after homogenization or lysis, the supernatant was centrifuged at 10000g for 10min for subsequent assays.
2) Adding 0.1mL of homogenate and PBS solution into a centrifuge tube or other appropriate container as blank control, adding 0.1mL of the standard substance with different concentrations for preparing a standard curve, and adding 0.1mL of sample for determination; subsequently, 0.2mL of MDA assay working solution was added. The concentration of the standard substance is as follows: 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:
TABLE 3 detection of the reaction System
| Item | Blank control | Standard article | Sample(s) |
| PBS | 0.1mL | - | - |
| Standard article | - | - | |
| Sample to be tested | - | - | 0.1mL |
| MDA reaction working solution | 0.2mL | 0.2mL | 0.2mL |
3) Mixing, and heating in boiling water bath for 15min. If the heating block is used for heating, the centrifugal tube cover is tightly pressed by a weight;
4) The mixture was 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, followed by measurement of absorbance at 532nm with a microplate reader.
2.20 kit for detecting content of GSH-Px in mouse brain black matter area
1) Preheating a microplate reader for 30min, adjusting the wavelength to 412nm, and adjusting the distilled water to zero;
2) Diluting the 80 mu mol/mL standard substance to 0.08 mu mol/mL;
3) Reagents were added as per table 4.
TABLE 4 kinds and amounts of reagents
Mixing, centrifuging at 4000rpm at room temperature for 5min, and collecting supernatant.
TABLE 5 types and amounts of reagents
| Diluent liquid | - | - | - | 100 |
| Supernatant fluid | 100 | 100 | - | - |
| Standard liquid | - | - | 100 | - |
| |
100 | 100 | 100 | 100 |
| Reagent five | 25 | 25 | 25 | 25 |
Mixing, standing at room temperature for 15min, and measuring absorbance at 412 nm.
2.21LC-MS/MS
Standard solution preparation ceramide and sphingomyelin standards were dissolved in chloroform/methanol (2/1,v/v) solvent, respectively, to make 1. Mu. Mo1/mL stock solutions. The stock solution was diluted with mobile phase A/B (1/1,v/v) to different concentrations of standard use solutions 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 as an internal standard was added at a concentration of 125pmol/mL, respectively.
Chromatographic conditions
Liquid chromatography column: agilent RX-SIL (2.1 mm. Times.100mm, 1.8 μm); mobile phase A: chloroform/methanol/aqueous 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 given in the following table: flow rate: 0.3mL/min; the column temperature was kept at 25 ℃; the sample size was 5. Mu.L. Gradient elution procedure 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 |
Conditions of Mass Spectrometry
The Agilent triple quadrupole mass spectrometer firstly adopts a parent ion scanning mode to screen out parent ions, and then adopts an MRM mode to quantify. The operating parameters are as follows: flow rate of drying gas: 9L/min; temperature of the drying gas: 300 ℃; atomizing gas pressure: 40psi.
2.22 data analysis
Data were analyzed and plotted using Graphpad Prism 5 (Version 5.01) and Adobe Illustrator CS6 (Version 16.0.0) for collation. All data are expressed as means ± SD, statistical differences between groups were tested by one-way ANOVA and Tukey's, and significant differences were considered for P values less than 0.05.
3. Results of the experiment
3.1 As shown in FIG. 8, FIG. 8 shows the effect of LA-1 on PD mouse behaviours, specifically: c57BL6 mice were injected with MPTP (30 mg/kg i.v.) for 5 days, followed by LA-1 (8.04 mg/kg i.v.) for 10 days. A in fig. 8 is total distance statistics, B in fig. 8 is center crossing number statistics, C in fig. 8 is center distance statistics, and D in fig. 8 is feeding statistics; and detecting by using an open field test. And (E) testing the rod. Results are expressed as mean ± standard deviation (n = 6). * p <0.05, p <0.01.
In an open field experiment, compared with a Control group, the total moving distance, the center crossing times, the center moving distance and the standing 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 erecting times of the mice in the MPTP + LA-1 group are obviously increased; in the pole climbing experiment, compared with the Control group, the pole climbing time of the MPTP group mouse is obviously increased, and compared with the MPTP group, the pole climbing time of the MPTP + LA-1 group mouse is obviously reduced.
3.2 As shown in FIG. 9, FIG. 9 shows the effect of LA-1 on TH expression in SN tissue in PD mice. The method comprises the following specific steps: c57BL6 mice were injected with MPTP (30 mg/kg i.v.) for 5 days, followed by LA-1 (8.04 mg/kg i.v.) for 10 days. Immunohistochemistry detected SN (Bar =50 μm) mouse TH, with results as a and B in fig. 9. Results are expressed as mean ± standard deviation (n = 6). * p <0.05, p <0.01.
In an immunohistochemical experiment, compared with a Control group, the average optical density of a TH positive area of a substantia nigra compact part of an MPTP group mouse is obviously reduced; compared with the MPTP group, the average light density of the TH positive area of the substantia nigra compacta of mice of the MPTP + LA-1 group is obviously improved.
3.3 As shown in FIG. 10, FIG. 10 shows the effect of LA-1 on SN expression in PD mice. The method specifically comprises the following steps: c57BL6 mice were injected with MPTP (30 mg/kg i.v.) for 5 days, followed by LA-1 (8.04 mg/kg i.v.) for 10 days. The percentage of TUNEL positive cells was calculated by TUNEL staining (Bar =50 μm) with results as a in figure 10 and results as B in figure 10. Results are expressed as mean ± SD (n = 6). * p <0.05, p <0.01.
In the TUNEL experiment, compared with the Control group, the number of positive cells of TUNEL in the substantia nigra of mice in the MPTP group is obviously increased; compared with the MPTP group, the number of the TUNEL positive cells in the substantia nigra of mice in the MPTP + LA-1 group 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. 11, FIG. 11 shows the effect of LA-1 on SYN expression in SN tissues of PD mice. The method specifically 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 i.v. drip) for 10 consecutive days. Expression of SYN in mouse SN was detected by confocal microscopy (a in fig. 11) (Bar =50 μm), mean fluorescence intensity (B in fig. 11), expressed as mean ± SD (n = 6). * p <0.05, p <0.01.
In an immunofluorescence experiment, compared with a Control group, the average fluorescence intensity of a black SYN positive area of an MPTP group mouse is obviously increased; compared with the MPTP group, the average fluorescence intensity of the SYN positive area of the substantia nigra of mice in the MPTP + LA-1 group is obviously reduced.
3.5 As shown in FIG. 12, FIG. 12 shows the effect of LA-1 on the expression of PSD-95 in SN tissue from PD mice. The method specifically 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 i.p.) for 10 consecutive days. Confocal microscopy (a in fig. 12) (Bar =50 μm) detected the expression of PSD-95 in mouse SN, the mean fluorescence intensity (B in fig. 12). Results are expressed as mean ± standard deviation (n = 6). * p <0.05, p <0.01.
In an immunofluorescence experiment, compared with a Control group, the average fluorescence intensity of a positive area of MPTP group mouse substantia nigra PSD-95 is obviously improved; compared with the MPTP group, the average fluorescence intensity of the positive area of the black PSD-95 of mice in the MPTP + LA-1 group is obviously reduced.
3.6 As shown in FIG. 13, FIG. 13 shows the effect of LA-1 on the expression of SN tissues DA, DOPAC, HVA, 5-HT, IL-1 β and TNF- α in PD mice, which is specifically: c57BL6 mice were injected with MPTP (30 mg/kg i.v.) for 5 days, followed by LA-1 (8.04 mg/kg i.v.) for 10 days. SN mice DA (A in FIG. 13), DOPAC (B in FIG. 13), HVA (C in FIG. 13), 5-HT (D in FIG. 13), IL-1 β (E in FIG. 13), TNF- α (F in FIG. 13) were assayed by ELISA. Results are expressed as mean ± standard deviation (n = 6). * p <0.05, p <0.01.
Compared with the Control group, the contents of substantia nigra DA, DOPAC, HVA and 5-HT of mice in the MPTP group are obviously reduced, and the contents of IL-1beta and TNF-alpha are obviously increased; compared with the MPTP group, the contents of the substantia nigra DA, the DOPAC, the HVA and the 5-HT of mice in the MPTP + LA-1 group are obviously increased, and the contents of the IL-1beta and the TNF-alpha are obviously reduced.
3.7 As shown in FIG. 14, FIG. 14 shows the effect of LA-1 on the expression of SOD, MDA, and GSH-px in SN tissues of PD mice. The method specifically comprises the following steps: c57BL6 mice were injected with MPTP (30 mg/kg i.v.) for 5 days, followed by LA-1 (8.04 mg/kg i.v.) for 10 days. SOD (A in FIG. 14), MDA (B in FIG. 14), GSH-px (C in FIG. 14). Results are expressed as mean ± standard deviation (n = 6). * p <0.05, p <0.01.
Compared with the Control group, the MPTP group (model group) has obviously reduced black SOD and GSH-px activity and obviously increased MDA content; compared with the MPTP group, the activity of the substantia nigra SOD and the GSH-px of mice in the MPTP + LA-1 group is obviously improved, 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 concrete is as follows: c57BL6 mice were injected with MPTP (30 mg/kg i.v.) for 5 days, followed by LA-1 (8.04 mg/kg i.v.) for 10 days. LC-MS detected sphingomyelin (A in FIG. 14), 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 contents of the substantia nigra sphingomyelin and the ceramide of the MPTP group mouse are obviously increased; compared with the MPTP group, the contents of the sphingomyelin nigra and the ceramide of mice in the MPTP + LA-1 group are obviously reduced.
The literature "Loganin substentially amaroid molecules formulations, pathology and cognitive impact in amouse model of Alzheimer' 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.2021 Oct 23 (20): 23739-2323756.) discloses the evaluation of the activity of Loganin in 3xTg-AD (triple transgenic) mice, where Loganin is used at a dose of 20mg/kg; in the test example of the present invention, APP/PS1 AD mice were used, and the loganin acid derivative of the structure of formula 1 prepared in example 1 was used at a dose of 8.04mg/kg, which is 2.49 times less than the dose of the loganin acid derivative of the structure of formula 1 prepared in example 1 of the present invention. The APP/PS1 AD mice used in the test examples of the present invention are double transgenic 2xTg, which are different from the three transgenic 3xTg reported in the above documents in mutated genes, but the common characteristics of the model mice in the above documents and the model mice in the present invention are that the mutated APP genes both have AD characteristics, which cause the amyloid plaques to appear in the brain (see "comparative study of APP/PS1 double transgenic and APP/PS1/Tau three transgenic Alzheimer disease model mice [ D ]. Zhou Yi. Zhongnan university, 2012."). Therefore, the dose of the present invention using a loganin acid derivative effective for the treatment of model mice is reduced by 2.49 times compared to the dose of loganin used in the above documents.
The active results show that the loganin acid derivative with the structure shown in the formula 1 can protect or repair the damage of a nervous system, and has a good treatment effect prospect on the Parkinson nervous system degenerative diseases or diseases.
Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments belong to the protection scope of the present invention.
Claims (10)
1. A loganic acid derivative having the structure shown in formula 1:
2. the process for the preparation of a loganic acid derivative according to claim 1, comprising the steps of:
mixing the loganic acid, the compound with the structure shown in the formula 2, a coupling reagent and an organic solvent for coupling reaction to obtain a compound with the structure shown in the formula 3;
in a hydrogen atmosphere, mixing a compound with a structure shown in a formula 3, a palladium-carbon catalyst and an organic solvent for carrying out reduction deprotection reaction to obtain the loganic acid derivative with a structure shown in a formula 1.
3. The method according to claim 2, wherein the compound of formula 2 is prepared by a method comprising 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 to perform an amide condensation reaction to obtain a compound with a structure shown in a formula 6;
and (3) mixing the compound with the structure shown in the formula 6 and an organic solution of hydrogen chloride for carrying out amino deprotection reaction to obtain the compound with the structure shown in the formula 2.
4. The method according to claim 3, wherein the compound of formula 4 is prepared by a method comprising 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 base 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. The method according to claim 3, wherein the compound of formula 5 is prepared by a method comprising 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 base 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 and an organic solution of hydrogen chloride for carrying out 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 the loganic acid to the compound having the structure represented by formula 2 is 1 (1.9-2.2).
7. The method according to claim 3, wherein the molar ratio of the compound having the structure represented by formula 4 to the compound having the structure represented by formula 5 is 1 (1.1 to 1.3).
8. The 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 method according to claim 5, wherein the molar ratio of the compound having a structure represented by formula 10 to the compound having a structure represented by formula 11 is 1 (1.1 to 1.3).
10. Use of a loganic acid derivative according to claim 1 or a loganic acid derivative prepared by the preparation method according to any one of claims 2 to 9 in the preparation of a medicament for treating and/or preventing a parkinson's nervous system degenerative disease or disorder.
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